Goodpasture antigen binding protein

ABSTRACT

The present invention provides isolated nucleic acid sequences and expression vectors encoding the Goodpasture antigen binding protein (GPBP), substantially purified GPBP, antibodies against GPBP, and methods for detecting GPBP.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.10/270,837 filed Oct. 11, 2002, which is a divisional of U.S.application Ser. No. 09/512,563 filed Feb. 24, 2000, now U.S. Pat. No.6,579,969, which claims priority to U.S. Provisional Patent ApplicationSer. No. 60/121,483, filed Feb. 24, 1999.

STATEMENT OF GOVERNMENT RIGHTS

This work was supported in part by Grants SAL91/0513, SAF94/1051 andSAF97/0065 from the Plan Nacional I+D of the Comisión Interministerialde Ciencia Tecnología (CICYT, Spain), Grant 93/0343 from Fondo deInvestigaciones Sanitarias (FISss, Spain) and Grants GV-3166/95,GV-C-VS-21-118-96 from la Direcció General d'Ensenyaments Universitarisi Investigació (Comunitat Valenciana, Spain); therefore the State ofSpain may have rights in the invention.

FIELD OF THE INVENTION

The invention relates to the fields of protein kinases, automimmunedisease, apoptosis, and cancer.

BACKGROUND OF THE INVENTION

Goodpasture (GP) disease is an autoimmune disorder described only inhumans. In GP patients, autoantibodies against the non-collagenousC-terminal domain (NC1) of the type IV collagen α3 chain (“Goodpastureantigen”) cause a rapidly progressive glomerulonephritis and often lunghemorrhage, the two cardinal clinical manifestations of the GP syndrome(see 1 for review. The reference numbers in this section correspond toreference list of Example 1).

The idea that common pathogenic events exist at least for someautoimmune disorders is suggested by the significant number of patientsdisplaying more than one autoimmune disease, and also by the strong andcommon linkage that some of these diseases show to specific MHChaplotypes (31, 32). The experimental observation that the autoantigenis the leading moiety in autoimmunity and that a limited number ofself-components are autoantigenic (31), suggest that theseself-components share biological features with important consequences inself/non-self recognition by the immune system. One possibility is thattriggering events, by altering different but specific self-components,would result in abnormal antigen processing. In certain individualsexpressing a particular MHC specificity, the abnormal peptides could berecognized by non-tolerized T cells and trigger an immune response (1).

We have previously explored the GP antigen to identify biologicalfeatures of relevance in autoimmune pathogenesis. Since the NC1 domainis a highly conserved domain among species and between the differenttype IV collagen α chains (α1-α6) (2), the exclusive involvement of thehuman α3(IV)NC1 in a natural autoimmune response suggests that thisdomain has structural and/or biological peculiarities of pathogenicrelevance. Consistent with this, the N-terminus of the human antigen ishighly divergent, and it contains a unique five-residue motif (KRGDS⁹)that conforms to a functional phosphorylation site for type A proteinkinases (3, 4). Furthermore, the human α3 gene, but not the otherrelated human or homologous genes from other species, is alternativelyspliced and generates multiple transcripts also containing thephosphorylatable N-terminal region (5-7). Recent studies indicate thatthe phosphorylation of the N-terminus of the GP antigen bycAMP-dependent protein kinase is up regulated by the presence of thealternative products (see Example 3 below). Specific serinephosphorylation and pre-mRNA alternative splicing are also associatedwith the biology of other autoantigens including the acetylcholinereceptor and myelin basic protein (MBP) (4). The latter is suspected tobe the major antigen in multiple sclerosis (MS), another exclusivelyhuman autoimmune disease in which the immune system targets the whitematter of the central nervous system. GP disease and MS are humandisorders that display a strong association with the same HLA class IIhaplotype (HLA DRB1*1501)(32, 33). This, along with the recent report ofdeath by GP disease of an MS patient carrying this HLA specificity (34),supports the existence of common pathogenic events in these humandisorders.

Thus, specific serine/threonine phosphorylation may be a majorbiological difference between the human GP antigen, the GP antigens ofother species, and the homologous domains from the other human α(IV)chains, and might be important in pathogenesis (1, 4).

Therefore, the identification and isolation of the specificserine/threonine kinase that phosphorylates the N-terminal region of thehuman GP antigen would be very advantageous for the diagnosis andtreatment of GP syndrome, and possibly for other autoimmune disorders.

SUMMARY OF THE INVENTION

The present invention fulfills the need in the art for theidentification and isolation of a serine/threonine kinase thatspecifically binds to and phosphorylates the unique N-terminal region ofthe human GP antigen. In one aspect, the present invention providesnucleic acid sequences encoding various forms of the Goodpasture antigenbinding protein (GPBP), as well as recombinant expression vectorsoperatively linked to the GPBP-encoding sequences.

In another aspect, the present invention provides host cells that havebeen transfected with the recombinant expression vectors. In a furtheraspect, the present invention provides substantially purified GPBP andantibodies that selectively bind to GPBP. In still further aspect, theinvention provides methods for detecting the presence of GPBP or nucleicacids encoding GPBP.

In a further aspect, the present invention provides methods fordetecting the presence of an autoimmune condition or apoptosis, whichcomprises detecting an increase in the expression of GPBP in a tissuecompared to a control tissue.

In another aspect, the present invention provides methods andpharmaceutical compositions for treating an autoimmune disorder,apoptosis, or a tumor, comprising modifying the expression or activityof GPBP in a patient in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Nucleotide and derived amino acid sequences of n4′. The denotedstructural features are from 5′ to 3′ end: the cDNA present in theoriginal clone (HeLa1) (dotted box), which contains the PH homologydomain (in black) and the Ser-Xaa-Yaa repeat (in gray); the heptadrepeat of the predictable coiled-coil structure (open box) containingthe bipartite nuclear localization signal (in gray); and a serine-richdomain (filled gray box). The asterisks denote the positions of in framestop codons.

FIG. 2. Distribution of GPBP in human tissues (Northern blot) and ineukaryotic species (Southern blot). A random primed ³²P-labeled HeLa1cDNA probe was used to identify homologous messages in a Northern blotof poly(A⁺)RNA from the indicated human tissues (panel A) or in aSouthern blot of genomic DNA from the indicated eukaryotic species(panel B). Northern hybridization was performed under highly stringentconditions to detect perfect matching messages and at low stringency inthe Southern to allow the detection of messages with mismatches. Noappreciable differences in the quality and amount of each individualpoly A+ RNA was observed by denaturing gel electrophoresis or whenprobing a representative blot from the same lot with human β-actin cDNA.The numbers denote the position and the sizes in kb of the RNA or DNAmarkers used.

FIG. 3. Experimental determination of the translation start site. In(A), the two cDNAs present in pc-n4′ and pc-FLAG-n4′ plasmids used fortransient expression are represented as black lines. The relativeposition of the corresponding predicted (n4′) or engineered (FLAG-n4′)translation start site is indicated (Met). In (B), the extracts fromcontrol (−), pc-n4′(n4′) or pc-FLAG-n4′ (FLAG-n4′) transfected 293 cellswere subjected to SDS-PAGE under reducing conditions in 10% gels. Theseparated proteins were transferred to a PVDF membrane (Millipore) andblotted with the indicated antibodies. The numbers and bars indicate themolecular mass in kDa and the relative positions of the molecular weightmarkers, respectively.

FIG. 4. Characterization of rGPBP from yeast and 293 cells. In (A), 1 μg(lane 1) or 100 ng (lanes 2 and 3) of yeast rGPBP were analyzed byreducing SDS-PAGE in a 10% gel. The separated proteins were stained withCoomassie blue (lane 1) or transferred and blotted with anti-FLAGantibodies (lane 2) or Mab14, a monoclonal antibody against GPBP (lane3). In (B), the cell extracts from GPBP-expressing yeast were analyzedas in A and blotted with anti-FLAG (lane 1), anti-PSer (lane 2),anti-PThr (lane 3) or anti-PTyr (lane 4) monoclonal antibodiesrespectively. In (C), 200 ng of either yeast rGPBP (lane 1),dephosphorylated yeast rGPBP (lane 2) or 293 cells-derived rGPBP (lane3) were analyzed as in B with the indicated antibodies. In (D), similaramounts of H₃ ³²PO₄-labeled non-transfected (lanes 1), stable pc-n4′transfected (lanes 2) or transient pc-FLAG-n4′ expressing (lanes 3) 293cells were lysed, precipitated with the indicated antibodies andanalyzed by SDS-PAGE and autoradiography. The molecular weight markersare represented with numbers and bars as in FIG. 3. The arrows indicatethe position of the rGPBP.

FIG. 5. Recombinant GPBP contains a serine/threonine kinase thatspecifically phosphorylates the N-terminal region of the human GPantigen. To assess phosphorylation, approximately 200 ng of yeast rGPBPwas incubated with [γ]³²P-ATP in the absence (A and B) or presence of GPantigen-derived material (C). In (A), the mixture was subjected toreducing SDS-PAGE (10% gel) and autoradiographed. In (B), the mixturewas subjected to ³²P-phosphoamino acid analysis by two-dimensionalthin-layer chromatography. The dotted circles indicate the position ofninhydrin stained phosphoamino acids. In (C), the phosphorylationmixtures of the indicated GP-derived material were analyzed by SDS-PAGE(15% gel) and autoradiography (GPpep1 and GPpep1Ala⁹) orimmunoprecipitated with Mab 17, a monoclonal antibody that specificallyrecognize GP antigen from human and bovine origin, and analyzed bySDS-PAGE (12.5%) and autoradiography (rGP, GP). The relative positionsof rGPBP (A), rGP antigen and the native human and bovine GP antigens(C) are indicated by arrows. The numbers and bars refer to molecularweight markers as in previous Figures.

FIG. 6. In-blot renaturation of the serine/threonine kinase present inrGPBP. Five micrograms of rGPBP from yeast were in-blot renatured. Therecombinant material was specifically identified by anti-FLAG antibodies(lane 1) and the in situ ³²P-incorporation detected by autoradiography(lane 2). The numbers and bars refer to molecular weight markers as inprevious Figures. The arrow indicates the position of the 89 kDa rGPBPpolypeptide.

FIG. 7. Immunological localization of GPBP in human tissues. Rabbitserum against the N-terminal region of GPBP (1:50) was used to localizeGPBP in human tissues. The tissues shown are kidney (A) glomerulus (B),lung (C), alveolus (D), liver (E), brain (F), testis (G), adrenal gland(H), pancreas (I) and prostate (J). Similar results were obtained usinganti-GPBP affinity-purified antibodies or a pool of culture medium fromseven different GPBP-specific monoclonal antibodies (anti-GPBP Mabs 3,4, 5, 6, 8, 10 and 14). Rabbit pre-immune serum did not stain any tissuestructure in parallel control studies. Magnification was 40× except in Band D where it was 100×.

FIG. 8. GPBPΔ26 is a splicing variant of GPBP. (A) Total RNA from normalskeletal muscle was retrotranscribed using primer 53c and subsequentlysubjected to PCR with primers 18m-53c (lane 2) or 15m-62c (lane 4).Control amplifications of a plasmid containing GPBP cDNA using the samepairs of primers are shown in lanes 1 and 3. Numbers on the left andright refer to molecular weight in base pairs. The region missing in thenormal muscle transcript was identified and its nucleotide sequence(lower case) and deduced amino acid sequence (upper case) are shown in(B). A clone of genomic DNA comprising the cDNA region of interest wassequenced and its structure is drawn in (C), showing the location andrelative sizes of the 78-bp exon spliced out in GPBPΔ26 (black box),adjacent exons (gray boxes), and introns (lines). The size of bothintron and exons is given and the nucleotide sequence of intron-exonboundaries is presented, with consensus for 5′ and 3′ splice sites shownin bold case.

FIG. 9. Differential expression of GPBP and GPBPΔ26. Fragmentsrepresenting the 78-bp exon (GPBP) or flanking sequences common to bothisoforms (GPBP/GPBPΔ26) were ³²P-labeled and used to hybridize humantissue and tumor cell line Northern blots (CLONTECH). The membranes werefirst hybridized with GPBP-specific probe, stripped and then reanalyzedwith GPBP/GPBPΔ26 probe. Washing conditions were less stringent forGPBP-specific probe (0.1% SSPE, 37° C. or 55° C.) than for theGPBP/GPBPΔ26 (0.1% SSPE, 68° C.) to increase GPBP and GPBPΔ26 signalsrespectively. No detectable signal was obtained for the GPBP probe whenthe washing program was at 68° C. (not shown).

FIG. 10. GPBPΔ26 displays lower phosphorylating activity than GPBP. (A)Recombinantly-expressed, affinity-purified GPBP (rGPBP) (lanes 1) orrGPBPΔ26 (lanes 2) were subjected to SDS-PAGE under reducing conditionsand either Coomasie blue stained (2 μg per lane) or blotted (200 ng perlane) with monoclonal antibodies recognizing the FLAG sequence (α-FLAG)or GPBP/GPBPΔ6 (Mab14). (B) 200 ng of rGPBP (lanes 1) or rGPBPΔ26 (lanes2) were in vitro phosphorylated without substrate to assayauto-phosphorylation (left), or with 5 nmol GPpep1 to measuretrans-phosphorylation activity (right). An arrowhead indicates theposition of the peptide. (C) 3 μg of rGPBP (lane 1) or rGPBPΔ26 (lane 2)were in-blot renatured as described under Material and Methods. Thenumbers and bars indicate the molecular mass in kDa and the relativeposition of the molecular weight markers, respectively.

FIG. 11. rGPBP and rGPBPΔ26 form very active high molecular weightaggregates. About 300 μg of rGPBP (A) or rGPBPΔ26 (B) were subjected togel filtration HPLC as described under Material and Methods. Verticalarrowheads and numbers respectively indicate the elution profile andmolecular mass (kDa) of the molecular weight standards used. Largeraggregates eluted in the void volume (I), and the bulk of the materialpresent in the samples eluted in the fractionation range of the columnas a second peak between the 669 and 158 kDa markers (II). Fifteenmicroliters of the indicated minute fractions were subjected to SDS-PAGEand Coomasie blue staining. Five microliters of the same fractions werein vitro phosphorylated as described in Materials and Methods, and thereaction stopped by boiling in SDS sample buffer. The fractions wereloaded onto SDS-PAGE, transferred to PVDF and autoradiographed for 1 or2 hours using Kodak X-Omat films and blotted using anti-FLAG monoclonalantibodies (Sigma).

FIG. 12. Self-interaction of GPBP and GPBPΔ26 assessed by a yeasttwo-hybrid system. (A) Cell transfected for the indicated combinationsof plasmids were selected on leucine-tryptophan-deficient medium (-Trp,-Leu), and independent transformants restreaked onto histidine-deficientplates (-Trp, -Leu, -His) in the presence or absence of 1 mM3-amino-triazole (3-AT), to assess interaction. The picture was taken 3days after streaking. (B) The bars represent mean values inβ-galactosidase arbitrary units of four independent β-galactosidasein-solution assays.

FIG. 13. GPBP is expressed associated with endothelial and glomerularbasement membranes. Paraffin embedded sections of human muscle (A) orrenal cortex (B, C) were probed with GPBP-specific antibodies (A,B) orwith Mab189, a monoclonal antibody specific for the human α3(IV)NC1 (C).Frozen sections of human kidney (D-F) were probed with Mab17, amonoclonal antibody specific for the α3(IV)NC1 domain (D), GPBP-specificantibodies (E), or sera from a GP patient (F). Control sera (chickenpre-immune and human control) did not display tissue-binding in parallelstudies (not shown).

FIG. 14. GPBP is expressed in human but not in bovine and murine renalcortex. Cortex from human (A, D), bovine (B, E) or murine (C, F) kidneywere paraffin embedded and probed with either GPBP-specific antibodies(A-C) or GPBP/GPBPΔ26-specific antibodies (D-F).

FIG. 15. GPBP is highly expressed in several autoimmune conditions.Skeletal muscle total RNA from a control individual (lane 1) or from aGP patient (lane 2) was subjected to RT-PCR as in FIG. 8, using theoligonucleotides 15m and 62c in the amplification program. Frozen (B-D)or paraffin embedded (E-G) human control skin (B, E) or skin affected bySLE (C, F) or lichen planus (D, G) were probed with GPBP-specificantibodies.

FIG. 16. Phosphorylation of GP alternative splicing products by PKA. Inleft panel, equimolecular amounts of rGP (lanes 1), rGPΔV (lanes 2),rGPΔIII (lanes 3) or rGPΔIII/IV/V (lanes 4), equivalent to 500 ng of theGP were phosphorylated at the indicated ATP concentrations. One-fifth ofthe total phosphorylation reaction mixture was separated by gelelectrophoresis and transferred to PVDF, autoradiographed (shown) andthe proteins blotted with M3/1, a specific monoclonal antibodyrecognizing all four species (shown) or using antibodies specific foreach individual C-terminal region (not shown). Arrowheads indicate theposition of each recombinant protein, from top to bottom, GP, GPΔV and,GPΔIII -GPΔIII/IV/V which displayed the same mobilities. Right panel:purified α3(IV)NC1 domain or hexamer was phosphorylated with PKA and 0.1μM ATP in the absence (lanes 1) or in the presence of 10 nmol ofpeptides representing the C-terminal region of either GPΔIII (lanes 2)or GPΔIII/IV/V (lanes 3). Where indicated the phosphorylation mixturesof purified α3(IV)NC1 domain were V8 digested and immunoprecipitatedwith antibodies specific for the N terminus of the human α3(IV)NC1domain (3). Bars and numbers indicate the position and sizes (kDa) ofthe molecular weight markers.

FIG. 17. Sequence alignment of GPΔIII and MBP. The phosphorylation sitesfor PKA (boxed) and the structural similarity for the sites at Ser 8 and9 of MBP and GPΔIII respectively are shown (underlined). The identity(vertical bars) and chemical homology (dots) of the corresponding exonII (bent arrow) of both molecular species are indicated. The completesequence of GPΔIII from the collagenase cleavage site (72-residues) isaligned with the 69-N terminal residues of MBP comprising the exon I andten residues of the exon II.

FIG. 18. Phosphorylation of recombinant MBP proteins by PKA. About 200ng of rMBP (lane 1), or Ser to Ala mutants thereof in position 8 (lane2) or 57 (lane 3), or rMPBΔII (lane 4) or Ser to Ala mutants thereof inposition 8 (lane 5) or 57 (lane 6), were phosphorylated by PKA and 0.1μM ATP. The mixtures were subjected to SDS-PAGE, transferred to PVDF andautoradiographed (Phosphorylation) and the individual molecular speciesblotted with monoclonal antibodies against human MBP obtained from RocheMolecular Biochemicals (Western).

FIG. 19. Phosphorylation of recombinant MBP proteins by GPBP. About 200ng of rMBP (lane 1), or Ser to Ala mutants thereof in positions 8 (lane2) or 57 (lane 3), or rMPBΔII (lane 4), or Ser to Ala mutants thereof inpositions 8 (lane 5) or 57 (lane 6), were subjected to SDS-PAGE,transferred to PVDF, and the area containing the proteins visualizedwith Ponceau and stripped out. The immobilized proteins were in situphosphorylated with rGPBP as described in Materials and Methods,autoradiographed (Phosphorylation) and subsequently blotted as in FIG.18 (Western).

FIG. 20. Regulation of the GPBP by the C terminal region of GPΔIII.About 200 ng of rGPBP were in vitro phosphorylated with 150 μM ATP inthe absence (lane 1) or in the presence of 5 nmol of GPΔIII-derivedpeptide synthesized either using Boc-(lane 2) or Fmoc-(lane 3)chemistry. The reaction mixtures were subjected to SDS-PAGE, transferredto PVDF and autoradiographed to asses autophosphorylation, andsubsequently blotted with anti-FLAG monoclonal antibodies (Sigma) todetermine the amount of recombinant material present (Western).

DETAILED DESCRIPTION OF THE INVENTION

All references cited are herein incorporated by reference in theirentirety.

The abbreviations used herein are: bp, base pair; DTT, dithiothreitol;DMEM, Dulbecco's modified Eagle's medium; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid; GP, Goodpasture; rGPΔIII, rGPΔIII/IV/V andrGPΔV, recombinant material representing the alternative forms of theGoodpasture antigen resulting from splicing out exon III, exon III, IVand V or exon V, respectively; GPBP and rGPBP, native and recombinantGoodpasture antigen binding protein; GPBPΔ26 and rGPBPΔ26, native andrecombinant alternative form of the GPBP; GST, glutathioneS-transferase; HLA, human lymphocyte antigens; HPLC, high performanceliquid chromatography; Kb, thousand base pairs; kDa, thousand daltons;MBP, rMBP, native and recombinant 21 kDa myelin basic protein; MBPΔIIand rMBPΔII, native and recombinant 18.5 kDa myelin basic protein thatresults from splicing out exon II; MBPΔV and MBPΔII/V, myelin basicprotein alternative forms resulting from splicing out exon V and exonsII and V, respectively; MHC, major histocompatibility complex; NC1,non-collagenous domain; PH, pleckstrin homology; PKA, cAMP-dependentprotein kinase; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodiumdodecylsulfate polyacrylamide gel electrophoresis; TBS, tris bufferedsaline.

Within this application, unless otherwise stated, the techniquesutilized may be found in any of several well-known references such as:Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells. A Manual of BasicTechnique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray,The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog(Ambion, Austin, Tex.).

As used herein, the term “GPBP” refers to Goodpasture binding protein,and includes both monomers and oligomers thereof. Human (SEQ ID NO:2),mouse (SEQ ID NO:4), and bovine GPBP sequences (SEQ ID NO:6) areprovided herein.

As used herein, the term “GPBPΔ26” refers to Goodpasture binding proteindeleted for the 26 amino acid sequence shown in SEQ ID NO:14, andincludes both monomers and oligomers thereof. Human (SEQ ID NO:8), mouse(SEQ ID NO:10), and bovine GPBP sequences (SEQ ID NO:12) are providedherein.

As used herein the term “GPBPpep1” refers to the 26 amino acid peptideshown in SEQ ID NO:14, and includes both monomers and oligomers thereof.

As used herein, the term “GP antigen” refers to the α3 NC1 domain oftype IV collagen.

As used herein, “MBP” refers to myelin basic protein.

In one aspect, the present invention provides isolated nucleic acidsthat encode GPBP, GPBPΔ26, and GPBPpep1, and mutants or fragmentsthereof. In one embodiment, the isolated nucleic acids comprisesequences substantially similar to SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or SEQ IDNO:25, or fragments thereof

In another aspect, the present invention provides isolated nucleic acidsthat encode alternative products of the GP antigen or MBP. In oneembodiment, the isolated nucleic acids comprise sequences that encodepeptides substantially similar to SEQ ID NO:43 and SEQ ID NO:44.

The phrase “substantially similar” is used herein in reference to thenucleotide sequence of DNA or RNA, or the amino acid sequence ofprotein, having one or more conservative or non-conservative variationsfrom the disclosed sequences, including but not limited to deletions,additions, or substitutions, wherein the resulting nucleic acid and/oramino acid sequence is functionally equivalent to the sequencesdisclosed herein. Functionally equivalent sequences will function insubstantially the same manner to produce substantially the same proteindisclosed herein. For example, functionally equivalent DNAs encodeproteins that are the same as those disclosed herein or that have one ormore conservative amino acid variations, such as substitution of anon-polar residue for another non-polar residue or a charged residue fora similarly charged residue. These changes include those recognized bythose of skill in the art as substitutions that do not substantiallyalter the tertiary structure of the protein.

In practice, the term substantially similar means that DNA encoding twoproteins hybridize to one another under conditions of moderate to highstringency, and encode proteins that have either the same sequence ofamino acids, or have changes in sequence that do not alter theirstructure or function. As used herein, substantially similar sequencesof nucleotides or amino acids share at least about 70% identity, morepreferably at least about 80% identity, and most preferably at leastabout 90% identity. It is recognized, however, that proteins (and DNA ormRNA encoding such proteins) containing less than the above-describedlevel of homology arising as splice variants or that are modified byconservative amino acid substitutions (or substitution of degeneratecodons) are contemplated to be within the scope of the presentinvention.

Stringency of hybridization is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(T_(M)) of the hybrids. T_(M) decreases approximately 1-1.5° C. withevery 1% decrease in sequence homology. In general, the stability of ahybrid is a function of sodium ion concentration and temperature.Typically, the hybridization reaction is performed under conditions oflower stringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions. Thus, as used herein, moderate stringency refers toconditions that permit hybridization of those nucleic acid sequencesthat form stable hybrids in 0.1% SSPE at 37° C. or 55° C., while highstringency refers to conditions that permit hybridization of thosenucleic acid sequences that form stable hybrids in 0.1% SSPE at 65° C.It is understood that these conditions may be duplicated using a varietyof buffers and temperatures and that they are not necessarily precise.Denhardt's solution and SSPE (see, e.g., Sambrook, Fritsch, andManiatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, 1989) are well known to those of skill in the art, asare other suitable hybridization buffers.

The isolated nucleic acid sequence may comprise an RNA, a cDNA, or agenomic clone with one or more introns. The isolated sequence mayfurther comprise additional sequences useful for promoting expressionand/or purification of the encoded protein, including but not limited topolyA sequences, modified Kozak sequences, and sequences encodingepitope tags, export signals, and secretory signals, nuclearlocalization signals, and plasma membrane localization signals.

In another aspect, the present invention provides recombinant expressionvectors comprising nucleic acid sequences that express GPBP, GPBPΔ26, orGPBPpep1, and mutants or fragments thereof. In one embodiment, thevectors comprise nucleic acid sequences that are substantially similarto the sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25, orfragments thereof.

In another aspect, the present invention provides recombinant expressionvectors comprising nucleic acid sequences that express peptides that aresubstantially similar to the amino acid sequence shown in SEQ ID NO:43,SEQ ID NO:44, or peptide fragments thereof.

“Recombinant expression vector” includes vectors that operatively link anucleic acid coding region or gene to any promoter capable of effectingexpression of the gene product. The promoter sequence used to driveexpression of the disclosed nucleic acid sequences in a mammalian systemmay be constitutive (driven by any of a variety of promoters, includingbut not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven byany of a number of inducible promoters including, but not limited to,tetracycline, ecdysone, steroid-responsive). The construction ofexpression vectors for use in transfecting prokaryotic cells is alsowell known in the art, and thus can be accomplished via standardtechniques. (See, for example, Sambrook, Fritsch, and Maniatis, in:Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989; Gene Transfer and Expression Protocols, pp.109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.)

The expression vector must be replicable in the host organisms either asan episome or by integration into host chromosomal DNA. In a preferredembodiment, the expression vector comprises a plasmid. However, theinvention is intended to include other expression vectors that serveequivalent functions, such as viral vectors.

In a further aspect, the present invention provides host cells that havebeen transfected with the recombinant expression vectors disclosedherein, wherein the host cells can be either prokaryotic or eukaryotic.The cells can be transiently or stably transfected. Such transfection ofexpression vectors into prokaryotic and eukaryotic cells can beaccomplished via any technique known in the art, including but notlimited to standard bacterial transformations, calcium phosphateco-precipitation, electroporation, or liposome mediated-, DEAE dextranmediated-, polycationic mediated-, or viral mediated transfection. (See,for example, Molecular Cloning. A Laboratory Manual (Sambrook, et al.,1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: AManual of Basic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc.New York, N.Y.),

In a still further aspect, the present invention provides substantiallypurified GPBP, GPBPΔ26, and GPBPpep1, and mutants or fragments thereof.In one embodiment, the amino acid sequence of the substantially purifiedprotein is substantially similar to SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, orpeptide fragments thereof.

In another aspect, the present invention provides substantially purifiedalternative products of the GP antigen and MBP. In one embodiment, theamino acid sequence of the substantially purified polypeptide issubstantially similar to SEQ ID NO:43, SEQ ID NO:44, or peptidefragments thereof.

As used herein, the term “substantially purified” means that the proteinhas been separated from its in vivo cellular environments. Thus, theprotein can either be purified from natural sources, or recombinantprotein can be purified from the transfected host cells disclosed above.In a preferred embodiment, the proteins are produced by the transfectedcells disclosed above, and purified using standard techniques. (See forexample, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press.)) The protein can thus be purifiedfrom prokaryotic or eukaryotic sources. In various further preferredembodiments, the protein is purified from bacterial, yeast, or mammaliancells.

The protein may comprise additional sequences useful for promotingpurification of the protein, such as epitope tags and transport signals.Examples of such epitope tags include, but are not limited to FLAG(Sigma Chemical, St. Louis, Mo.), myc (9E10) (Invitrogen, Carlsbad,Calif.), 6-His (Invitrogen; Novagen, Madison, Wis.), and HA (BoehringerManheim Biochemicals). Examples of such transport signals include, butare not limited to, export signals, secretory signals, nuclearlocalization signals, and plasma membrane localization signals.

In another aspect, the present invention provides antibodies thatselectively bind to GPBP, GPBPΔ26, or GPBPpep1. In one aspect, theantibodies selectively bind to a protein comprising a sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or peptide fragmentsthereof. Such antibodies can be produced by immunization of a hostanimal with either the complete GPBP, or with antigenic peptidesthereof. The antibodies can be either polyclonal or monoclonal.

In another aspect, the present invention provides antibodies thatselectively bind to a polypeptide comprising an amino acid sequencesubstantially similar to a sequence selected from the group consistingof SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ IDNO:50, SEQ ID NO:54, or antigenic fragments thereof. The antibodies canbe either polyclonal or monoclonal.

Antibodies can be made by well-known methods, such as described inHarlow and Lane, Antibodies; A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., (1988). In one example, preimmuneserum is collected prior to the first immunization. Substantiallypurified proteins of the invention, or antigenic fragments thereof,together with an appropriate adjuvant, is injected into an animal in anamount and at intervals sufficient to elicit an immune response. Animalsare bled at regular intervals, preferably weekly, to determine antibodytiter. The animals may or may not receive booster injections followingthe initial immunization. At about 7 days after each boosterimmunization, or about weekly after a single immunization, the animalsare bled, the serum collected, and aliquots are stored at about −20° C.Polyclonal antibodies against the proteins and peptides of the inventioncan then be purified directly by passing serum collected from the animalthrough a column to which non-antigen-related proteins prepared from thesame expression system without GPBP-related proteins bound.

Monoclonal antibodies can be produced by obtaining spleen cells from theanimal. (See Kohler and Milstein, Nature 256, 495-497 (1975)). In oneexample, monoclonal antibodies (mAb) of interest are prepared byimmunizing inbred mice with the proteins or peptides of the invention,or an antigenic fragment thereof. The mice are immunized by the IP or SCroute in an amount and at intervals sufficient to elicit an immuneresponse. The mice receive an initial immunization on day 0 and arerested for about 3 to about 30 weeks. Immunized mice are given one ormore booster immunizations of by the intravenous (IV) route.Lymphocytes, from antibody positive mice are obtained by removingspleens from immunized mice by standard procedures known in the art.Hybridoma cells are produced by mixing the splenic lymphocytes with anappropriate fusion partner under conditions which will allow theformation of stable hybridomas. The antibody producing cells and fusionpartner cells are fused in polyethylene glycol at concentrations fromabout 30% to about 50%. Fused hybridoma cells are selected by growth inhypoxanthine, thymidine and aminopterin supplemented Dulbecco's ModifiedEagles Medium (DMEM) by procedures known in the art. Supernatant fluidsare collected from growth positive wells and are screened for antibodyproduction by an immunoassay such as solid phase immunoradioassay.Hybridoma cells from antibody positive wells are cloned by a techniquesuch as the soft agar technique of MacPherson, Soft Agar Techniques, inTissue Culture Methods and Applications, Kruse and Paterson, Eds.,Academic Press, 1973.

To generate such an antibody response, the proteins of the presentinvention are typically formulated with a pharmaceutically acceptablecarrier for parenteral administration. Such acceptable adjuvantsinclude, but are not limited to, Freund's complete, Freund's incomplete,alum-precipitate, water in oil emulsion containing Corynebacteriumparvum and tRNA. The formulation of such compositions, including theconcentration of the polypeptide and the selection of the vehicle andother components, is within the skill of the art.

The term antibody as used herein is intended to include antibodyfragments thereof which are selectively reactive with the proteins andpeptides of the invention, or fragments thereof. Antibodies can befragmented using conventional techniques, and the fragments screened forutility in the same manner as described above for whole antibodies. Forexample, F(ab′)₂ fragments can be generated by treating antibody withpepsin. The resulting F(ab′)₂ fragment can be treated to reducedisulfide bridges to produce Fab′ fragments.

In a further aspect, the invention provides methods for detecting thepresence of the proteins or peptides of the invention in a proteinsample, comprising providing a protein sample to be screened, contactingthe protein sample to be screened with an antibody against the proteinsor peptides of the invention, and detecting the formation ofantibody-antigen complexes. The antibody can be either polyclonal ormonoclonal as described above, although monoclonal antibodies arepreferred. As used herein, the term “protein sample” refers to anysample that may contain the proteins or peptides of the invention, andfragments thereof, including but not limited to tissues and portionsthereof, tissue sections, intact cells, cell extracts, purified orpartially purified protein samples, bodily fluids, nucleic acidexpression libraries. Accordingly, this aspect of the present inventionmay be used to test for the presence of GPBP, GPBPΔ26, GPBPpep1, oralternative products of the GP antigen in these various protein samplesby standard techniques including, but not limited to,immunolocalization, immunofluorescence analysis, Western blot analysis,ELISAs, and nucleic acid expression library screening, (See for example,Sambrook et al, 1989.) In one embodiment, the techniques may determineonly the presence or absence of the protein or peptide of interest.Alternatively, the techniques may be quantitative, and provideinformation about the relative amount of the protein or peptide ofinterest in the sample. For quantitative purposes, ELISAs are preferred.

Detection of immunocomplex formation between the proteins or peptides ofthe invention, or fragments thereof, and their antibodies or fragmentsthereof, can be accomplished by standard detection techniques. Forexample, detection of immunocomplexes can be accomplished by usinglabeled antibodies or secondary antibodies. Such methods, including thechoice of label are known to those ordinarily skilled in the art.(Harlow and Lane, Supra). Alternatively, the polyclonal or monoclonalantibodies can be coupled to a detectable substance. The term “coupled”is used to mean that the detectable substance is physically linked tothe antibody. Suitable detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase. Examples of suitable prosthetic-group complexesinclude streptavidin/biotin and avidin/biotin. Examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin. An example of a luminescent material includesluminol. Examples of suitable radioactive material include ¹²⁵I, ¹³¹I,³⁵S or ³H.

Such methods of detection are useful for a variety of purposes,including but not limited to detecting an autoimmune condition,identifying cells targeted for or undergoing apoptosis,immunolocalization of the proteins of interest in a tissue sample,Western blot analysis, and screening of expression libraries to findrelated proteins.

In yet another aspect, the invention provides methods for detecting thepresence in a sample of nucleic acid sequences encoding the GPBP,GPBPΔ26, GPBPpep1, or alternative products of the GP antigen comprisingproviding a nucleic acid sample to be screened, contacting the samplewith a nucleic acid probe derived from the isolated nucleic acidsequences of the invention, or fragments thereof, and detecting complexformation.

As used herein, the term “sample” refers to any sample that may containGPBP-related nucleic acid, including but not limited to tissues andportions thereof, tissue sections, intact cells, cell extracts, purifiedor partially purified nucleic acid samples, DNA libraries, and bodilyfluids. Accordingly, this aspect of the present invention may be used totest for the presence of GPBP mRNA or DNA in these various samples bystandard techniques including, but not limited to, in situhybridization, Northern blotting, Southern blotting, DNA libraryscreening, polymerase chain reaction (PCR) or reverse transcription-PCR(RT-PCR). (See for example, Sambrook et al, 1989.) In one embodiment,the techniques may determine only the presence or absence of the nucleicacid of interest. Alternatively, the techniques may be quantitative, andprovide information about the relative amount of the nucleic acid ofinterest in the sample. For quantitative purposes, quantitative PCR andRT-PCR are preferred. Thus, in one example, RNA is isolated from asample, and contacted with an oligonucleotide derived from the nucleicacid sequence of interest, together with reverse transcriptase undersuitable buffer and temperature conditions to produce cDNAs from theGPBP-related RNA. The cDNA is then subjected to PCR using primer pairsderived from the nucleic acid sequence of interest. In a preferredembodiment, the primers are designed to detect the presence of the RNAexpression product of SEQ ID NO:5, and the amount of GPBP geneexpression in the sample is compared to the level in a control sample.

For detecting the nucleic acid sequence of interest, standard labelingtechniques can be used to label the probe, the nucleic acid of interest,or the complex between the probe and the nucleic acid of interest,including, but not limited to radio-, enzyme-, chemiluminescent-, oravidin or biotin-labeling techniques, all of which are well known in theart. (See, for example, Molecular Cloning: A Laboratory Manual(Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), GeneExpression Technology (Methods in Enzymology, Vol. 185, edited by D.Goeddel, 1991. Academic Press, San Diego, Calif.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.)).

Such methods of nucleic acid detection are useful for a variety ofpurposes, including but not limited to diagnosing an autoimmunecondition, identifying cells targeted for or undergoing apoptosis, insitu hybridization, Northern and Southern blot analysis, and DNA libraryscreening.

As demonstrated in the following examples, GPBP shows preferentialexpression in tissue structures that are commonly targeted innaturally-occurring automimmune responses, and is highly expressed inseveral autoimmune conditions, including but not limited to GoodpastureSyndrome (GP), systemic lupus erythematosus (SLE), and lichen planus.Furthermore, following a similar experimental approach to that describedbelow, recombinant proteins representing autoantigens in GP disease (α3Type IV collagen), SLE (P1 ribosomal phosphoprotein and Sm-D1 smallnuclear ribonucleoproteins) and dermatomyositis (hystididyl-tRNAsynthetase) were shown to be in vitro substrates of GPBP.

Thus, in a preferred embodiment, detection of GPBP expression is used todetect an autoimmune condition. A sample that is being tested iscompared to a control sample for the expression of GPBP, wherein anincreased level of GPBP expression indicates the presence of anautoimmune condition. In this embodiment, it is preferable to useantibodies that selectively bind to GPBPpep1, which is present in GPBPbut not in GPBPΔ26.

Furthermore, as shown in the accompanying examples, GPBP isdown-regulated in tumor cell lines, and the data suggest thatGPBP/GPBPΔ26 are likely to be involved in cell signaling pathways thatinduce apoptosis, which may be up-regulated during autoimmunepathogenesis and down-regulated during cell transformation to preventautoimmune attack to transformed cells during tumor growth. Thus, thedetection methods disclosed herein can be used to detect cells that aretargeted for, or are undergoing apoptosis.

In another aspect, the present invention provides a method for treatingan autoimmune disorder, a tumor, or for preventing cell apoptosiscomprising modification of the expression or activity of GPBP, GPBPΔ26,or a protein comprising a polypeptide substantially similarly toGPBPpep1 in a patient in need thereof. Modifying the expression oractivity of GPBP, GPBPΔ26, or a protein comprising a polypeptidesubstantially similarly to GPBPpep1 can be accomplished by usingspecific inducers or inhibitors of GPBP expression or activity, GPBPantibodies, gene or protein therapy using GP or myelin basic proteinalternative products, cell therapy using host cells expressing GP ormyelin basic protein alternative products, antisense therapy, or othertechniques known in the art. In a preferred embodiment, the methodfurther comprises administering a substantially purified alternativeproduct of the GP antigen or MBP to modify the expression or activity ofGPBP, GPBPΔ26, or a protein comprising a polypeptide substantiallysimilarly to GPBPpep1. As used herein, “modification of expression oractivity” refers to modifying expression or activity of either the RNAor protein product.

In a further aspect, the present invention provides pharmaceuticalcompositions, comprising an amount effective of substantially purifiedalternative products of the GP antigen or MBP to modify the expressionor activity of GPBP RNA or protein, and a pharmaceutically acceptablecarrier.

For administration, the active agent is ordinarily combined with one ormore adjuvants appropriate for the indicated route of administration.The compounds may be mixed with lactose, sucrose, starch powder,cellulose esters of alkanoic acids, stearic acid, talc, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine,and/or polyvinyl alcohol, and tableted or encapsulated for conventionaladministration. Alternatively, the compounds of this invention may bedissolved in saline, water, polyethylene glycol, propylene glycol,carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanutoil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.Other adjuvants and modes of administration are well known in thepharmaceutical art. The carrier or diluent may include time delaymaterial, such as glyceryl monostearate or glyceryl distearate alone orwith a wax, or other materials well known in the art.

The present invention may be better understood with reference to theaccompanying examples that are intended for purposes of illustrationonly and should not be construed to limit the scope of the invention, asdefined by the claims appended hereto.

EXAMPLE 1 Characterization of GPBP

Here we report the cloning and characterization of a novel type ofserine/threonine kinase that specifically binds to and phosphorylatesthe unique N-terminal region of the human GP antigen.

Materials and Methods

Synthetic polymers-Peptides. GPpep1, KGKRGDSGSPATWTTRGFVFT (SEQ IDNO:26), representing residues 3-23 of the human GP antigen andGPpep1Ala⁹, KGKRGDAGSPATWTTRGFVFT (SEQ ID NO:27), a mutant Ser⁹ to Ala⁹thereof, were synthesized by MedProbe and CHIRON. FLAG peptide, was fromSigma.

Oligonucleotides. The following as well as several other GPBP-specificoligonucleotides were synthesized by Genosys and GIBCO BRL: ON-GPBP-54m:TCGAATTCACCATGGCCCCACTAGCCGACTACAA (SEQ ID NO:28) GGACGACGATGACAAG.ON-GPBP-55c: CCGAGCCCGACGAGTTCCAGCTCTGATTATCCGA (SEQ ID NO:29)CATCTTGTCATCGTCG. ON-HNC-B-N-14m: CGGGATCCGCTAGCTAAGCCAGGCAAGGATGG. (SEQID NO:30) ON-HNC-B-N-16c: CGGGATCCATGCATAAATAGCAGTTCTGCTGT. (SEQ IDNO:31)

Isolation and characterization of cDNA clones encoding humanGPBP-Several human λ-gt11 cDNA expression libraries (eye, fetal andadult lung, kidney and HeLa S3, from CLONTECH) were probed for cDNAsencoding proteins interacting with GPpep1. Nitrocellulose filters(Millipore) prepared following standard immunoscreening procedures wereblocked and incubated with 1-10 nmoles per ml of GPpep1 at 37° C.Specifically bound GPpep1 was detected using M3/1A monoclonal antibodies(7). A single clone was identified in the HeLa-derived library (HeLa1).Specificity of fusion protein binding was confirmed by similar bindingto recombinant eukaryotic human GP antigen. The EcoRI cDNA insert ofHeLa1 (0.5-kb) was used to further screen the same library and toisolate overlapping cDNAs. The largest cDNA (2.4-kb) containing theentire cDNA of HeLa1 (n4′) was fully sequenced.

Northern and Southern blots-Pre-made Northern and Southern blots(CLONTECH) were probed with HeLa1 cDNA following manufacturerinstructions.

Plasmid construction, expression and purification of recombinantproteins-GPBP-derived material. The original λ-gt11 HeLa1 clone wasexpressed as a lysogen in E. Coli Y1089 (8). The correspondingβ-galactosidase-derived fusion protein containing the N-terminal 150residues of GPBP was purified from the cell lysate using an APTG-agarosecolumn (Boehringer). The EcoRI 2.4-kb fragment of n4′ was subcloned inBluescribe M13+ vector (Stratagene) (BS-n4′), amplified and used forsubsequent cloning. A DNA fragment containing (from 5′ to 3′), an EcoRIrestriction site, a standard Kozak consensus for translation initiation,a region coding for a tag peptide sequence (FLAG, DYKDDDDK (SEQ IDNO:32)), and the sequence coding for the first eleven residues of GPBPincluding the predicted Met_(i) and a Ban II restriction site, wasobtained by hybridizing ON-GPBP-54m and ON-GPBP-55c, and extending withmodified T₇ DNA polymerase (Amersham). The resulting DNA product wasdigested with EcoRI and BanII, and ligated with the BanII/EcoRI cDNAfragment of BS-n4′ in the EcoRI site of pHIL-D2 (Invitrogen) to producepHIL-FLAG-n4′. This plasmid was used to obtain Mut^(s) transformants ofthe GS115 strain of Pichia pastoris and to express FLAG-taggedrecombinant GPBP (rGPBP) either by conventional liquid culture or byfermentation procedures (Pichia Expression Kit, Invitrogen). The celllysates were loaded onto an anti-FLAG M2 column (Sigma), the unboundmaterial washed out with Tris buffered saline (TBS, 50 mM Tris-HCl, pH7.4, 150 mM NaCl) or salt-supplemented TBS (up to 2M NaCl), and therecombinant material eluted with FLAG peptide. For expression incultured human kidney-derived 293 cells (ATCC 1573-CRL), the 2.4- or2.0-kb EcoRI cDNA insert of either BS-n4′ or pHIL-FLAG-n4′ was subclonedin pcDNA3 (Invitrogen) to produce pc-n4′ and pc-FLAG-n4′ respectively.When used for transient expression, 18 hours after transfection thecells were lysed with 3.5-4 μl/cm² of chilled lysis buffer (1% NonidetP-40 or Triton-X100, 5mM EDTA and 1 mM PMSF in TBS) with or without 0.1%SDS, depending on whether the lysate was to be used for SDS-PAGE orFLAG-purification, respectively. For FLAG purification, the lysate offour to six 175 cm² culture dishes was diluted up to 50 ml with lysisbuffer and purified as above. For stable expression, the cells weresimilarly transfected with pc-n4′ and selected for three weeks with 800μg/ml of G418. For bacterial recombinant expression, the 2.0-kb EcoRIcDNA fragment of pHIL-FLAG-n4′ was cloned in-frame downstream of theglutathione S-transferase (GST)-encoding cDNA of pGEX-5x-1 (Pharmacia).The resulting construct was used to express GST-GPBP fusion protein inDH5α cells (9).

GP antigen-derived material. Human recombinant GP antigen (rGP) wasproduced in 293 cells using the pRc/CMV-BM40 expression vectorcontaining the α3-specific cDNA between ON-HNC-B-N-14m andON-HNC-B-N-16c. The expression vector is a pRc/CMV (Invitrogen)-derivedvector provided by Billy G. Hudson (Kansas University Medical Center)that contains cDNA encoding an initiation Met, a BM40 signal peptidefollowed by a tag peptide sequence (FLAG), and a polylinker cloningsite. To obtain α3-specific cDNA, a polymerase chain reaction wasperformed using the oligonucleotides above and a plasmid containing thepreviously reported α3(IV) cDNA sequence (3) as template (clone C2). Forstable expression of rGP, 293 cells were transfected with the resultingconstruct (fα3VLC) and selected with 400 μg/ml of G418. The harvestedrGP was purified using an anti-FLAG M2 column.

All the constructs were verified by restriction mapping and nucleotidesequencing.

Cell culture and DNA transfection-Human 293 cells were grown inDulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetalcalf serum. Transfections were performed using the calcium phosphateprecipitation method of the Profection Mammalian Transfection Systems(Promega). Stably transfected cells were selected by their resistance toG418. Foci of surviving cells were isolated, cloned and amplified.

Antibody production-Polyclonal antibodies against the N-terminal regionof GPBP. Cells expressing HeLa1 λ-gt11 as a lysogen were lysed bysonication in the presence of Laemmli sample buffer and subjected toelectrophoresis in a 7.5% acrylamide preparative gel. The gel wasstained with Coomassie blue and the band containing the fusion proteinof interest excised and used for rabbit immunization (10). Theanti-serum was tested for reactivity using APTG-affinity purifiedantigen. To obtain affinity-purified antibodies, the anti-serum wasdiluted 1:5 with TBS and loaded onto a Sepharose 4B column containingcovalently bound affinity purified antigen. The bound material waseluted and, unless otherwise indicated, used in the immunochemicalstudies.

Monoclonal antibodies against GPBP. Monoclonal antibodies were producedessentially as previously reported (7) using GST-GPBP. The supernatantsof individual clones were analyzed for antibodies against rGPBP.

In vitro phosphorylation assays-About 200 ng of rGPBP were incubatedovernight at 30° C. in 25 mM β-glycerolphosphate (pH 7.0), 0.5 mM EDTA,0.5 mM EGTA, 8 mM MgCl₂, 5 mM MnCl₂, 1 mM DTT and 0.132 μM γ-³²P-ATP, inthe presence or absence of 0.5-1 μg of protein substrates or 10 nmolesof synthetic peptides, in a total volume of 50 μl.

In vivo phosphorylation assays-Individual wells of a 24-well dish wereseeded with normal or with stably pc-n4′ transfected 293 cells. When thecells were grown to the desired density, a number of wells of the normal293 cells were transfected with pc-FLAG-n4′. After 12 hours, the culturemedium was removed, 20 μCi/well of H₃ ³²PO4 in 100 μl of phosphate-freeDMEM added, and incubation continued for 4 hours. The cells were lysedwith 300 μl/well of TBS containing 1% Triton X-100, 2 mM EDTA, 1 mMPMSF, 50 mM NaF and 0.2 mM vanadate, and extracted with specificantibodies and Protein A-Sepharose. When anti-GPBP serum was used, thelysate was pre-cleared using pre-immune serum and Protein A-Sepharose.

In vitro dephosphorylation of rGPBP-About 1 μg of rGPBP wasdephosphorylated in 100 μl of 10 mM Tris-acetate (pH 7.5), 10 mMmagnesium acetate and 50 mM potassium acetate with 0.85 U of calfintestine alkaline phosphatase (Pharmacia) for 30 min at 30° C.

Renaturation assays-In-blot renaturation assays were performed using 1-5μg of rGPBP as previously described (11).

Nucleotide sequence analysis-cDNA sequence analyses were performed bythe dideoxy chain termination method using [α]³⁵S-dATP, modified T₇ DNApolymerase (Amersham) and universal or GPBP-specific primers (8-10).

³²P-Phosphoamino acid analysis-Immunopurified rGPBP or HPLCgel-filtration fractions thereof containing the material of interestwere phosphorylated, hydrolyzed and analyzed in one dimensional (4) ortwo dimensional thin layer chromatography (12). When performing twodimensional analysis, the buffer for the first dimension was formicacid:acetic acid:water (1:3.1:35.9) (pH 1.9) and the buffer for thesecond dimension was acetic acid:pyridine:water (2:0.2:37.8) (pH 3.5).Amino acids were revealed with ninhydrin, and ³²P-phosphoamino acids byautoradiography.

Physical methods and immunochemical techniques-SDS-PAGE andWestern-blotting were performed as in (4). Immunohistochemistry studieswere done on human multi-tissue control slides (Biomeda, Biogenex) usingthe ABC peroxidase method (13).

Computer analysis-Homology searches were carried out against the GenBankand SwissProt databases with the BLAST 2.0 (14) at the NCBI server, andagainst the TIGR Human Gene Index database for expressed sequence tags,using the Institute for Genomic Research server. The search forfunctional patterns and profiles was performed against the PROSITEdatabase using the ProfileScan program at the Swiss Institute ofBioinformatics (15). Prediction of coiled-coil structures was done atthe Swiss Institute for Experimental Cancer Research using the programCoils (16) with both 21 and 28 residue windows.

Results

Molecular cloning of GPBP-To search for proteins specificallyinteracting with the divergent N-terminal region of the human GPantigen, a 21-residue peptide (GPpep1; SEQ ID NO:26)), encompassing thisregion and flanking sequences, and specific monoclonal antibodiesagainst it were combined to screen several human cDNA expressionlibraries. More than 5×10⁶ phages were screened to identify a singleHeLa-derived recombinant encoding a fusion protein specificallyinteracting with GPpep1 without disturbing antibody binding.

Using the cDNA insert of the original clone (HeLa1), we isolated a2.4-kb cDNA (n4′) that contains 408-bp of 5′-untranslated sequence, anopen reading frame (ORF) of 1872-bp encoding 624 residues, and 109-bp of3′-untranslated sequence (FIG. 1) (SEQ ID NO:1-2). Other structuralfeatures are of interest. First, the predicted polypeptide (hereinafterreferred to as GPBP) has a large number of phosphorylatable (17.9%) andacidic (16%) residues unequally distributed along the sequence. Serine,which is the most abundant residue (9.3%), shows preference for twoshort regions of the protein, where it comprises nearly 40% of the aminoacids, compared to an average of less than 7% throughout the rest of thepolypeptide chain. It is also noteworthy that the more N-terminal,serine-rich region consists mainly of a Ser-Xaa-Yaa repeat. Acidicresidues are preferentially located at the N-terminal three-quarters ofthe polypeptide, with nearly 18% of the residues being acidic. Theseresidues represent only 9% in the most C-terminal quarter of thepolypeptide, resulting in a polypeptide chain with two electricallyopposite domains. At the N-terminus, the polypeptide contains apleckstrin homology (PH) domain, which has been implicated in therecruitment of many signaling proteins to the cell membrane where theyexert their biological activities (17). Finally, a bipartite nucleartargeting sequence (18) exists as an integral part of a heptad repeatregion that meets all the structural requirements to form a coiled-coil(16).

Protein data bank searches revealed homologies almost exclusively withinthe approximately 100 residues at the N-terminal region harboring the PHdomain. The PH domain of the oxysterol-binding protein is the mostsimilar, with an overall identity of 33.5% and a similarity of 65.2%with GPBP. In addition, the Caenorhabditis elegans cosmid F25H2(accession number Q93569) contains a hypothetical ORF that displays anoverall identity of 26.5% and a similarity of 61% throughout the entireprotein sequence, indicating that similar proteins are present in lowerinvertebrates. Several human expressed sequence tags (accession numbersAA287878, AA287561, AA307431, AA331618, AA040134, AA158618, AA040087,AA122226, AA158617, AA121104, AA412432, AA412433, AA282679 and N27578)possess a high degree of nucleotide identity (above 98%) with thecorresponding stretches of the GPBP cDNA, suggesting that they representhuman GPBP. Interestingly, the AA287878 EST shows a gap of 67nucleotides within the sequence corresponding to the GPBP5′-untranslated region, suggesting that the GPBP pre-mRNA isalternatively spliced in human tissues (not shown).

The distribution and expression of the GPBP gene in human tissues wasfirst assessed by Northern blot analysis (FIG. 2, panel A). The gene isexpressed as two major mRNAs species between 4.4-kb and 7.5-kb in lengthand other minor species of shorter lengths. The structural relationshipbetween these multiple mRNA species is not known and their relativeexpression varies between tissues. The highest expression level is seenin striated muscle (skeletal and heart), while lung and liver show thelowest expression levels.

Southern blot studies analysis of genomic DNA from different speciesindicated that homologous genes exist throughout phylogeny (FIG. 2,panel B). Consistent with the human origin of the probe, thehybridization intensities decreased in a progressive fashion as theorigin of the genomic DNA moves away from humans in evolution.

Experimental determination of the translation start site-Toexperimentally confirm the predicted ORF, eukaryotic expression vectorscontaining either the 2.4-kb of cDNA of n4′, or only the predicted ORFtagged with a FLAG sequence (FIG. 3A), were used for transientexpression assays in 293 cells. The corresponding extracts were analyzedby immunoblot using GPBP- or FLAG-specific antibodies. The GPBP-specificantibodies bind to a similar major polypeptide in both transfectedcells, but only the polypeptide produced by the engineered constructexpressed the FLAG sequence (FIG. 3B). This located the translationstart site of the n4′ cDNA at the predicted Met and confirmed theproposed primary structure. Furthermore, the recombinant polypeptidesdisplayed a molecular mass higher than expected (80 versus 71 kDa)suggesting that GPBP undergoes post-translational modifications.

Expression and characterization of yeast rGPBP-Yeast expression andFLAG-based affinity-purification were combined to produce rGPBP (FIG.4A). A major polypeptide of ˜89 kDa, along with multiple relatedproducts displaying lower M_(r), were obtained. The recombinant materialwas recognized by both anti-FLAG and GPBP-specific antibodies,guaranteeing the fidelity of the expression system. Again, however, theM_(r) displayed by the major product was notably higher than predictedand even higher than the M_(r) of the 293 cell-derived recombinantmaterial, supporting the idea that GPBP undergoes important anddifferential post-translational modifications. Since phosphorylatableresidues are abundant in the polypeptide chain, we investigated theexistence of phosphoamino acids in the recombinant materials. By usingmonoclonal or polyclonal (not shown) antibodies against phosphoserine(Pser), phosphothreonine (PThr) and phosphotyrosine (PTyr), weidentified the presence of all three phosphoresidues either in yeastrGPBP (FIG. 4B) or in 293 cell-derived material (not shown). Thespecificity of the antibodies was further assessed by partiallyinhibiting their binding by the addition of 5-10 mM of the correspondingphosphoamino acid (not shown). This suggests that the phosphoresiduecontent varies depending upon the cell expression system, and that theM_(r) differences are mainly due to phosphorylation. Dephosphorylatedyeast-derived material consistently displayed similar M_(r) to thematerial derived from 293 cells, and phosphoamino acid contentcorrelates with SDS-PAGE mobilities (FIG. 4C). As an in vivomeasurement, the phosphorylation of rGPBP in the 293 cells was assessed(FIG. 4D). Control cells (lanes 1) and cells expressing rGPBP in astable (lanes 2) or transient (lanes 3) mode were cultured in thepresence of H₃ ³²PO₄. Immunoprecipitated recombinant material contained³²p, indicating that phosphorylation of GPBP occurred in vivo andtherefore is likely to be a physiological process.

The rGPBP is a serine/threonine kinase that phosphorylates theN-terminal region of the human GP antigen-Although GPBP does not containthe conserved structural regions required to define the classiccatalytic domain for a protein kinase, the recent identification andcharacterization of novel non-conventional protein kinases (19-27)encouraged the investigation of its phosphorylating activity. Additionof [y³²P]ATP to rGPBP (either from yeast or 293 cells (not shown)) inthe presence of Mn²⁺ and Mg²⁺ resulted in the incorporation of ³²p asPSer and PThr in the major and related products recognized by bothanti-FLAG and specific antibodies (FIG. 5A and B), indicating that theaffinity-purified material contains a Ser/Thr protein kinase. To furthercharacterize this activity, GPpep1, GPpep1Ala⁹ (a GPpep1 mutant withSer⁹ replaced by Ala), native and recombinant human GP antigens, andnative bovine GP antigen were assayed (FIG. 5C). Affinity-purified rGPBPphosphorylates all human-derived material to a different extent.However, in similar conditions, no appreciable ³²P-incorporation wasobserved in the bovine-derived substrate. The lower ³²p incorporationdisplayed by GPpep1Ala⁹ when compared with GPpep1, and the lack ofphosphorylation of the bovine antigen, indicates that the kinase presentin rGPBP discriminates between human and bovine antigens, and that Ser⁹is a target for the kinase.

Although the purification system provides high quality material, thepresence of contaminants with a protein kinase activity could not beruled out. The existence of contaminants was also suggested by thepresence of a FLAG-containing 40 kDa polypeptide, which displayed noreactivity with specific antibodies nor incorporation of ³²p in thephosphorylation assays (FIG. 4A and 5A). To precisely identify thepolypeptide harboring the protein kinase activity, we performed in vitrokinase renaturation assays after SDS-PAGE and Western-blotted (FIG. 6).We successfully combined the use of specific antibodies (lane 1) andautoradiographic detection of in situ ³²P-incorporation (lane 2), andidentified the 89 kDa rGPBP material as the primary polypeptideharboring the Ser/Thr kinase activity. The lack of ³²P-incorporation inthe rGPBP-derived products, as well as in the 40 kDa contaminant,further supports the specificity of the renaturation assays and locatesthe kinase activity to the 89 kDa polypeptide. Recently, it has beenshown that traces of protein kinases intimately associated with apolypeptide can be released from the blot membrane, bind to, andphosphorylate the polypeptide during the labeling step (28). To assessthis possibility in our system, we performed renaturation studies usinga small piece of membrane containing the 89 kDa polypeptide, eitheralone or together with membrane pieces representing the differentregions of the blot lane. We observed similar ³²P-incorporation at the89 kDa polypeptide regardless of the co-incubated pieces (not shown),indicating that if there are co-purified protein kinases in our samplethey are not phosphorylating the 89 kDa polypeptide in the renaturationassays unless they co-migrate. Co-migration does not appear to be aconcern, however, since rGPBP deletion mutants (GPBPΔ26 and R3; seebelow) displaying different mobilities also have kinase activities andcould be similarly in-blot renatured (not shown).

Immunohistochemical localization of the novel kinase-To investigate GPBPexpression in human tissues we performed immunohistochemical studiesusing specific polyclonal (FIG. 7) or monoclonal antibodies (not shown).Although GPBP is widely expressed in human tissues, it shows tissue andcell-specificity. In kidney, the major expression is found at the tubuleepithelial cells and the glomerular mesangial cells and podocytes. Atthe lung alveolus, the antibodies display a linear pattern suggestive ofa basement membrane localization, along with staining of pneumocytes.Liver shows low expression in the parenchyma, but high expression inbiliary ducts. Expression in the central nervous system is observed inthe white matter, but not in the neurons of the brain. In testis, a highexpression in the spermatogonium contrasts with the lack of expressionin Sertoli cells. The adrenal gland shows a higher level of expressionin cortical cells versus the medullar. In the pancreas, GPBP ispreferentially expressed in Langerhans islets versus the exocrinemoiety. In prostate, GPBP is expressed in the epithelial cells but notin the stroma (FIG. 7). Other locations with high expression of GPBP arestriated muscle, epithelial cells of intestinal tract, and Purkinjecells of the cerebellum (not shown). In general, in tissues where GPBPis highly expressed the staining pattern is mainly diffuse cytosolic.However in certain locations there is, in addition, an importantstaining reinforcement at the nucleus (spermatogonium), at the plasmamembrane (pneumocyte, hepatocyte, prostate epithelial cells, whitematter) or at the extracellular matrix (alveolus) (FIG. 7).

Discussion

Our data show that GPBP is a novel, non-conventional serine/threoninekinase. We also present evidence that GPBP discriminates between humanand bovine GP antigens, and targets the phosphorylatable region of humanGP antigen in vitro. Several lines of evidence indicate that the 89 kDapolypeptide is the only kinase in the affinity purified rGPBP. First, wefound no differences in auto- or trans-phosphorylation among rGPBPsamples purified in the presence of 150 mM, 0.5 M, 1 M or 2 M salt (notshown), suggesting that rGPBP does not carry intimately bound kinases.Second, there is no FLAG-containing, yeast-derived kinase in oursamples, since material purified using GPBP-specific antibodies shows nodifferences in phosphorylation (not shown). Third, a deletion mutant(GPBPΔ26; see below) displays reduced auto- and trans-phosphorylationactivities (not shown), demonstrating that the 89 kD polypeptide is theonly portion of the rGPBP with the ability to carry out phosphatetransfer.

Although GPBP is not homologous to other non-conventional kinases, theyshare some structural features including an N-terminal a-helixcoiled-coil (26, 27), serine-rich motifs (24), high phosphoamino acidscontent (27), bipartite nuclear localization signal (27), and theabsence of a typical nucleotide or ATP binding motif (24, 27).

Immunohistochemistry studies show that GPBP is a cytosolic polypeptidealso found in the nucleus, associated with the plasma membrane andlikely at the extracellular matrix associated with the basementmembrane, indicating that it contains the structural requirements toreach all these destinations. The nuclear localization signal and the PHdomain confer to it the potential to reach the nucleus and the cellmembrane, respectively (17, 29, 30). Although GPBP does not contain thestructural requirements to be exported, the 5′-end untranslated regionof its mRNA includes an upstream ORF of 130 residues with an in-framestop codon at the beginning (FIG. 1). A mRNA editing process inserting asingle base pair (U) would generate an operative in-frame start site andan ORF of 754-residues containing an export signal immediatelydownstream of the edited Met (not shown). Polyclonal antibodies againsta synthetic peptide representing part of this hypotheticalextra-sequence (PRSARCQARRRRGGRTSS (SEQ ID NO:33)) display a linearvascular reactivity in human tissues suggestive of an extracellularbasement membrane localization (data not shown).

Alternatively, a splicing phenomenon could generate transcripts withadditional unidentified exon(s) that would provide the structuralrequirements for exportation. The multiple cellular localization, thehigh content in PTyr, and the lack of tyrosine kinase activity in vitro,suggest that GPBP is itself the target of specific tyrosine kinase(s)and therefore likely involved in specific signaling cascade(s).

As discussed above, specific serine phosphorylation, as well as pre-mRNAalternative splicing, are associated with the biology of severalautoantigens, including the GP antigen, acetylcholine receptor andmyelin basic protein (MBP) (4). The latter is suspected to be the majorantigen in multiple sclerosis (MS), another exclusively human autoimmunedisease in which the immune system targets the white matter of thecentral nervous system. GP disease and MS are human disorders thatdisplay a strong association with the same HLA class II haplotype (HLADRB1*1501)(32, 33). This, along with the recent report of death by GPdisease of a MS patient carrying this HLA specificity (34), supports theexistence of common pathogenic events in these human disorders.

Phosphorylation of specific serines has been shown to changeintracellular proteolysis (35-40). Conceivably, alterations in proteinphosphorylation can affect processing and peptide presentation, and thusmediate autoimmunity. GP antigen-derived peptide presentation by theHLA-DR15 depends more on processing than on preferences of relativelyindiscriminate DR15 molecules (41), suggesting that if processing isinfluenced by abnormal phosphorylation, the resulting peptides wouldlikely be presented by this HLA. Our more recent data indicate that inboth the GP and MBP systems, the production of alternative splicingproducts serves to regulate the phosphorylation of specific andstructurally homologous PKA sites, suggesting that this or a closelyrelated kinase is the in vivo phosphorylating enzyme. Alterations in thedegree of antigen phosphorylation, caused either by an imbalance inalternative products, or by the action of an intruding kinase thatderegulates phosphorylation of the same motifs, could lead to anautoimmune response in predisposed individuals. rGPBP phosphorylates thehuman GP antigen at a major PKA phosphorylation site in an apparentlyunregulated fashion, since the presence of specific alternative productsof the GP antigen did not affect phosphorylation of the primary antigenby GPBP (not shown).

Although GPBP is ubiquitously expressed, in certain organs and tissuesit shows a preference for cells and tissue structures that are target ofcommon autoimmune responses: the Langerhans cells (type I diabetes); thewhite matter of the central nervous system (multiple sclerosis); thebiliary ducts (primary biliary cirrhosis); the cortical cells of theadrenal gland (Addison disease); striated muscle cells (myastheniagravis); spermatogonium (male infertility); Purkinje cells of thecerebellum (paraneoplasic cerebellar degeneration syndrome); andintestinal epithelial cells (pernicious anemia, autoimmune gastritis andenteritis). All the above observations point to this novel kinase as anattractive candidate to be considered when envisioning a model for humanautoimmune disease.

References for the Background and Example 1

-   1 Saus, J. (1998) Goodpasture's Syndrome. Encyclopedia of    Immunology, 2nd Ed., Delves, P. J., and Roitt, I. M. Eds., Academic    Press Limited, London, UK-   2 Leinonen, A., Mariyama, M., Mochizuki, T., Tryggvason, K., and    Reeders, S. T. (1994) J. Biol. Chem. 269, 26172-26177-   3 Quinones, S., Bernal, D., García-Sogo, M., Elena, S. F., and    Saus, J. (1992) J. Biol. Chem. 267, 19780-19784-   4 Revert, F., Penadés J. R., Plana, M., Bernal, D., Johansson, C.,    Itarte, E., Cervera, J., Wieslander, J., Quinones, S., and    Saus, J. (1995) J. Biol. Chem. 270, 13254-13261-   5 Bernal, D., Quinones, S., and Saus, J. (1993) J. Biol. Chem. 268,    12090-12094-   6 Feng, L., Xia, Y., and Wilson, C. B. (1994) J. Biol. Chem. 269,    2342-2348-   7 Penadés, J. R., Bernal, D., Revert, F., Johansson, C.,    Fresquet, V. J., Cervera, J., Wieslander, J., Quinones, S., and    Saus, J. (1995) Eur. J. Biochem. 229, 754-760-   8 Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular    Cloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor    Laboratory, Cold Spring Harbor, N.Y.-   9 Coligan, J. E., Dunn, B. N., Ploegh, H. L., Speicher, D. W., and    Winfield, P. T. (1995-97) Current Protocols in Protein Science, John    Wiley & Sons Eds., New York, N.Y.-   10 Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D.,    Deidman, J. G., Smith, J. A., and Struhl, K. (1994-98) Current    Protocols in Molecular Biology, John Wiley & Sons Eds., New York,    N.Y.-   11 Ferrel, J. E., and Martin, G. S. (1991) Methods in Enzymology    200, 430-435-   12 Boyle, W. J., van der Geer, P., and Hunter, T. (1991) Methods in    Enzymology 201, 110-149-   13 Hsu, S. M., Raine, L., and Fanger, H. (1981) J. Histochem.    Cytochem. 29, 577-580-   14 Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J.,    Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res.    25, 3389-3402-   15 Bairoch, A., Bucher, P., and Hofmann, K. (1997) Nucleic Acids    Res. 25, 217-221-   16 Lupas, A. (1996) Trends Biochem. Sci. 21, 375-382-   17 Lemmon, M. A., Falasca, M., Ferguson, K. M., and    Schlessinger, J. (1997) Trends Cell Biol. 7, 237-242-   18 Boulikas, T. (1993) Crit. Rev. Eukaryot. Gene Expr. 3, 193-227-   19 Csermely, P., and Kahn, C. R. (1991) J. Biol. Chem. 266,    4943-4950-   20 Maru, Y., and Witte, O. N.(1991) Cell 67, 459-468-   21 Beeler, J. F., LaRochelle, W. J., Chedid, M., Tronick, S. R., and    Aaronson, S. A. (1994) Mol. Cell. Biol. 14, 982-988-   22 Csermely, P., Miyata, Y., Schnaider, T., and Yahara, I. (1995) J.    Biol. Chem. 270, 6381-6388-   23 Dikstein, R., Ruppert, S., and Tjian, R. (1996) Cell 84, 781-790-   24 Eichinger, L., Bomblies, L., Vandekerckhove, J., Schleicher, M.,    and Gettermans, J. (1996) EMBO J. 15, 5547-5556-   25 Côté, G. P., Luo, X., Murphy, M. B., and    Egelhoff, T. T. (1997) J. Biol. Chem. 272, 6846-6849-   26 Ryazanov, A. G., Ward, M. D., Mendola, C. E., Pavur, K. S.,    Dorovkov, M. V., Wiedmann, M., Erdjument-Bromage, H., Tempst, P.,    Parmer, T. G., Prostko, C. R., Germino, F. J., and    Hait, W. N. (1997) Proc. Natl. Acad. Sci. USA 94, 4884-4889-   27 Fraser, R. A., Heard, D. J., Adam, S., Lavigne, A. C., Le    Douarin, B., Tora, L., Losson, R., Rochette-Egly, C., and    Chambon, P. (1998) J. Biol. Chem. 273, 16199-16204-   28 Langelier, Y., Champoux, L., Hamel, M., Guilbault, C., Lamarche,    N., Gaudreau, P., and Massie, B.(1998) J. Biol. Chem. 273, 1435-1443-   29 Lemmon, M. A., and Ferguson, K. M. (1998) Curr. Top. Microbiol.    Immunol. 228, 39-74-   30 Rebecchi, M. J., and Scarlata, S. (1998) Annu. Rev. Biophys.    Biomol. Struct. 27, 503-528-   31 Roitt, I. (1994) Autoimmune diseases in Essential Immunology,    383-439, 8^(th) Ed., Blackwell Scientific, Oxford, UK-   32 Erlich, H., and Apple, R. (1998) MHC disease associations.    Encyclopedia of Immunology, 2nd Ed., Delves, P. J., and Roitt, I. M.    Eds., Academic Press Limited, London, UK-   33 Phelps, R. G., Turner, A. N., and Rees, A. J. (1996) J. Biol.    Chem. 271, 18549-18553-   34 Henderson, R. D., Saltissi, D., and Pender, M. P. (1998) Acta    Neurol. Scand. 98, 134-135-   35 Litersky, J. M., and Johnson, G. V. W. (1992) J. Biol. Chem. 267,    1563-1568.-   36 Brown, K., Gerstberger, S., Carlson, L., Franzoso, G., and    Siebenlist, U. (1995) Science 267, 1485-1488-   37 Chen, Z. J., Parent, L., and Maniatis, T. (1996) Cell 84, 853-862-   38 Aberle, H., Bauer, A., Stappert, J., Kispert, A., and    Kemler, R. (1997) EMBO J. 16, 3797-3804-   39 Regnier, C. H., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z.,    and Rothe, M. (1997) Cell 90, 373-383-   40 Vlach, J., Hennecke, S., and Amati, B. (1997) EMBO J. 16,    5334-5344-   41 Phelps, R. G., Jones, V. L., Coughlan, M., Turner, A. N., and    Rees, A. J. (1998) J. Biol. Chem. 273, 11440-11447

EXAMPLE 2 GPBP Alternative Splicing

Here we report the existence of two isoforms of GPBP that are generatedby alternative splicing of a 78-base pair (bp) long exon that encodes a26-residue serine-rich motif. Both isoforms, GPBP and GPBPΔ26, exist ashigh molecular aggregates that result from polypeptide self-aggregation.The presence of the 26-residue peptide in the polypeptide chain resultsin a molecular species that self-interacts more efficiently and formsaggregates with higher specific activity. Finally, we present evidencessupporting the observation that GPBP is implicated in human autoimmunepathogenesis.

Material and Methods.

Synthetic polymers:

Peptides. GPpep1, KGKRGDSGSPATWTTRGFVFT (SEQ ID NO:26), is described inExample 1. GPBPpep1, PYSRSSSMSSIDLVSASDDVHRFSSQ (SEQ ID NO:14),representing residues 371-396 of GPBP was synthesized by Genosys.

Oligonucleotides. The following oligonucleotides were synthesized byLife Technologies, Inc., 5′ to 3′: ON-GPBP-11m, G CGG GAC TCA GCG GCCGGA TTT TCT (SEQ ID NO:34); ON-GPBP-15m, AC AGC TGG CAG AAG AGA C (SEQID NO:35); ON-GPBP-20c, C ATG GGT AGC TTT TAA AG (SEQ ID NO; 36);ON-GPBP-22m, TA GAA GAA CAG TCA CAG AGT GAA AAG G (SEQ ID NO;37);ON-GPBP-53c, GAATTC GAA CAA AAT AGG CTT TC (SEQ ID NO:38); ON-GPBP-56m,CCC TAT AGT CGC TCT TC (SEQ ID NO:39); ON-GPBP-57c, CTG GGA GCT GAA TCTGT (SEQ ID NO:40); ON-GPBP-62c, GTG GTT CTG CAC CAT CTC TTC AAC (SEQ IDNO:41); ON-GPBP-A26, CA CAT AGA TTT GTC CAA AAG GTT GAA GAG ATG GTG CAGAAC (SEQ ID NO:42).

Reverse transcriptase and polymerase chain rection (RT-PCR). Total RNAwas prepared from different control and GP tissues as described in (15).Five micrograms of total RNA was retrotranscribed using Ready-To-GoYou-Prime First-Strand beads (Amersham Pharmacia Biotech) and 40 pmol ofON-GPBP-53c. The corresponding cDNA was subjected to PCR using the pairsof primers ON-GPBP-11m/ON-GPBP-53c or ON-GPBP-15m/ON-GPBP-62c. Theidentity of the products obtained with 15m-62c was further confirmed byAlu I restriction. To specifically amplify GPBP transcripts, PCR wasperformed using primers ON-GPBP-15m/ON-GPBP-57c.

Northern hybridization studies. Pre-made human multiple-tissue and tumorcell-line Northern Blots (CLONTECH) were probed with a cDNA containingthe 78-bp exon present only in GPBP or with a cDNA representing bothisoforms. The corresponding cDNAs were obtained by PCR using the pair ofprimers ON-GPBP-56m and ON-GPBP-57c using GPBP as a template, or withprimers ON-GPBP-22m and ON-GPBP-20c, using GPBPΔ26 as a template. Theresulting products were random-labeled and hybridized following themanufacturers' instructions.

Plasmid construction, expression and purification of recombinantproteins. The plasmid pHIL-FLAG-n4′, used for recombinant expression ofFLAG-tagged GPBP in Pichia pastoris has been described elsewhere (4).The sequence coding for the 78-bp exon was deleted by site-directedmutagenesis using ON-GPBP-Δ26 to generate the plasmid pHIL-FLAG-n4′Δ26.Expression and affinity-purification of recombinant GPBP and GPBPΔ26 wasdone as in (4).

Gel-filtration HPLC. Samples of 250 μl were injected into a gelfiltration PE-TSK-G4000SW HPLC column equilibrated with 50 mM Tris-HClpH 7.5, 150 mM NaCl. The material was eluted from the column at 0.5ml/min, monitored at 220 nm and minute fractions collected.

In vitro phosphorylation assays. The auto-, trans-phosphorylation andin-blot renaturation studies were performed as in Example 1.

Antibodies and immunochemical techniques. Polyclonal antibodies wereraised by in chicken against a synthetic peptide (GPBPpep1) representingthe sequence coded by the 78-bp exon (Genosys). Egg yolks were diluted1:10 in water, the pH adjusted to 5.0. After 6 hours at 4° C., thesolution was clarified by centrifugation (25 min at 10000×g at 4° C.)and the antibodies precipitated by adding 20% (w/v) of sodium sulfate at20.000×g, 20′. The pellets were dissolved in PBS (1 ml per yolk) andused for immunohistochemical studies. The production of antibodiesagainst GPBP/GPBPΔ26 or against α3(IV)NC1 domain are discussed above(see also 4, 13).

Sedimentation velocity. Determination of sedimentation velocities wereperformed in an Optima XL-A analytical ultracentrifuge (BeckmanInstruments Inc.), equipped with a VIS-UV scanner, using a Ti60 rotorand double sector cells of Epon-charcoal of 12 mm optical path-length.Samples of ca. 400 μl were centrifuged at 30,000 rpm at 20° C. andradial scans at 220 nm were taken every 5 min. The sedimentationcoefficients were obtained from the rate of movement of the soluteboundary using the program XLAVEL (supplied by Beckman).

Sedimentation equilibrium. Sedimentation equilibrium experiments weredone as described above for velocity experiments with samples of 70 μl,and centrifuged at 8,000 rpm. The experimental concentration gradientsat equilibrium were analyzed using the program EQASSOC (Beckman) todetermine the corresponding weight average molecular mass. A partialspecific volumes of 0.711 cm³/g for GPBP and 0.729 cm³/g for GPBPΔ26were calculated from the corresponding amino acid compositions.

Physical methods and immunochemical techniques. SDS-PAGE and Westernblotting were performed under reducing conditions as previouslydescribed (3). Immunohistochemistry studies were done on formalin fixedparaffin embedded tissues using the ABC peroxidase method (4) or onfrozen human biopsies fixed with cold acetone using standard proceduresfor indirect immunofluorescence.

Two hybrid studies. Self-interaction studies were carried out inSaccharomyces cerevisiae (HF7c) using pGBT9 and pGAD424 (CLONTECH) togenerate GAL4 binding and activation domain-fusion proteins,respectively. Interaction was assessed following the manufacture'srecommendations. β-galactosidase activity was assayed with X-GAL (0.75mg/ml) for in situ and with ortho-nitrophenyl β-D galactopyranoside(0.64 mg/ml) for the in-solution determinations.

Results

Identification of two spliced GPBP variants. To characterize the GPBPspecies in normal human tissues, we coupled reverse transcription to apolymerase chain reaction (RT-PCR) on total RNA from different tissues,using specific oligonucleotides that flank the full open reading frameof GPBP. A single cDNA fragment displaying lower size than expected wasobtained from skeletal muscle-derived RNA (FIG. 8A), and from kidney,lung, skin, or adrenal gland-derived RNA (not shown). By combiningnested PCR re-amplifications and endonuclease restriction mapping, wedetermined that all the RT-PCR products corresponded to the samemolecular species (not shown). We fully sequenced the 2.2-Kb of cDNAfrom human muscle and found it identical to HeLa-derived material exceptfor the absence of 78-nucleotides (positions 1519-1596), which encode a26-residues motif (amino acids 371-396) (FIG. 8B). We therefore namedthis more common isoform of GPBP as GPBPΔ26.

To investigate whether the 78-bp represent an exon skipped transcriptduring pre-mRNA processing, we used this cDNA fragment to probe ahuman-derived genomic library and we isolated a ˜14-Kb clone. Bycombining Southern blot hybridization and PCR, the genomic clone wascharacterized and a contiguous DNA fragment of 12482-bp was fullysequenced (SEQ ID 25). The sequence contained (from 5′ to 3′), 767-bp ofintron sequence, a 93-bp exon, an 818-bp intron, the 78-bp exon sequenceof interest, a 9650-bp intron, a 96-bp exon and a 980-bp intron sequence(FIG. 8C). The exon-intron boundaries determined by comparing thecorresponding DNA and cDNA sequences meet the canonical consensus for 5′and 3′ splice sites (FIG. 8C) (5), thus confirming the exon nature ofthe 78-bp sequence. The GPBP gene was localized to chromosome 5q13 byfluorescence in situ hybridization (FISH) using the genomic clone as aprobe (not shown).

The relative expression of GPBP in human-derived specimens was assessedby Northern blot analysis, using either the 78-bp exon or a 260-bp cDNArepresenting the flanking sequence of 78-bp (103-bp 5′ and 157-bp 3′)present in both GPBP and GPBPΔ26 (FIG. 9). The 78-bp containing themolecular species of interest were preferably expressed in striatedmuscle (both skeletal and heart) and brain, and poorly expressed inplacenta, lung and liver. In contrast to GPBPΔ26, the GPBP was expressedat very low levels in kidney, pancreas and cancer cell lines.

All the above indicates that GPBP is expressed at low levels in normalhuman tissues, and that the initial lack of detection by RT-PCR of GPBPcan be attributed to a preferential amplification of the more abundantGPBPΔ26. Indeed, the cDNA of GPBP could be amplified from human tissues(skeletal muscle, lung, kidney, skin and adrenal gland) when thespecific RT-PCR amplifications were done using 78-bp exon-specificoligonucleotides (not shown). This also suggests that GPBPΔ26 mRNA isthe major transcript detected in Northern blot studies when using thecDNA probe representing both GPBP and GPBPΔ26.

Recombinant expression and functional characterization of GPBPΔ26. Toinvestigate whether the absence of the 26-residue serine-rich motifwould affect the biochemical properties of GPBP, we expressed andpurified both isoforms (rGPBP and rGPBPΔ26), and assessed their auto-and trans-phosphorylation activities (FIG. 10). As reported above forrGPBP (see also 4), rGPBPΔ26 is purified as a single major polypeptideand several related minor products (FIG. 10 A). However, the number andrelative amounts of the derived products vary compared to rGPBP, andthey display M_(r) on SDS-PAGE that cannot be attributed simply to the26-residue deletion. This suggests that the 26-residue motif hasimportant structural and functional consequences that could account forthe reduced in-solution auto- and trans-phosphorylation activitiesdisplayed by rGPBPΔ26 (FIG. 10B). Interestingly, the differences inspecific activity shown in the in-solution assays were not evident whenautophosphorylation was assessed in-blot after SDS-PAGE andrenaturation, suggesting that the 26-residue motif likely has importantfunctional consequences at the quaternary structure level. Renaturationstudies further showed that phosphate transfer activities reside in themajor polypeptides representing the proposed open reading frames, andare not detectable in derived minor products.

rGPBP and rGPBP-26 exist as very active high molecular weightaggregates. Gel filtration analysis of affinity-purified rGPBP orrGPBPΔ26 yielded two chromatographic peaks (I and II), both displayinghigher MW than expected for the individual molecular species, asdetermined by SDS-PAGE studies (89 kDa and 84 kDa, respectively ) (FIG.11). The bulk of the recombinant material eluted as a single peakbetween the 158 kDa and the 669 kDa molecular weight markers (peak II),while limited amounts of rGPBP and only traces of rGPBPΔ26 eluted inpeak I (>1000 kDa). Aliquots of fractions representing eachchromatographic profile were subjected to SDS-PAGE and stained, orincubated in the presence of ³²p[γ] ATP, and analyzed by immunoblot andautoradiography. Along with the major primary polypeptide, everychromatographic peak contained multiple derived products of higher orlower sizes indicating that the primary polypeptide associates to formhigh molecular weight aggregates that are stabilized by covalent andnon-covalent bonds (not shown). The kinase activity also exhibited twopeaks coinciding with the chromatographic profiles. However, peak Ishowed a much higher specific activity than peak II, indicating thatthese high molecular weight aggregates contained a much more active formof the kinase. Equal volumes of rGPBP fractions number 13 and 20exhibited comparable phosphorylating activity, even though the proteincontent is approximately 20 times lower in fraction 13, as estimated byWestern blot and Coomasie blue staining (FIG. 11A). The specificactivities of rGPBP and rGPBPΔ26 at peak II are also different, and areconsistent with the studies shown for the whole material, thussupporting the hypothesis that the presence of the 26-rediue serine-richmotif renders a more active kinase. These results also suggest that bothrGPBP and rGPBPΔ26 exist as oligomers under native conditions, and thatboth high molecular weight aggregate formation and specific activity aregreatly dependent on the presence of the 26-residue serine-rich motif.Analytical centrifugation analysis of rGPBP revealed that peak Icontained large aggregates (over 10⁷ Da). Peak II of rGPBP contained ahomogenous population of 220±10 kDa aggregates, likely representingtrimers with a sedimentation coefficient of 11S. Peak II of rGPBPΔ26however consisted of a more heterogenous population that likely containsseveral oligomeric species. The main population (ca. 80%) displayed aweight average molecular mass of 310±10 kDa and a coefficient ofsedimentation of 14S.

GPBP and GPBPΔ26 self-interact in a yeast two-hybrid system. To assessthe physiological relevance of the self-aggregation, and to determinethe role of the 26-residue motif, we performed comparative studies usinga two-hybrid interaction system in yeast. In this type of study, thepolypeptides whose interaction is under study are expressed as a part ofa fusion protein containing either the activation or the binding domainsof the transcriptional factor GAL4. An effective interaction between thetwo fusion proteins through the polypeptide under study would result inthe reconstitution of the transcriptional activator and the subsequentexpression of the two reporter genes, Lac Z and His3, allowing colonycolor detection and growth in a His-defective medium, respectively. Weestimated the intensity of interactions by the growth-rate inhistidine-defective medium, in the presence of different concentrationsof a competitive inhibitor of the His3 gene product (3-AT), and aquantitative colorimetric liquid β-galactosidase assay. A representativeexperiment is presented in FIG. 12. When assaying GPBPΔ26 forself-interaction, a significant induction of the reporter genes wasobserved, while no expression was detectable when each fusion proteinwas expressed alone or with control fusion proteins. The insertion ofthe 26-residue motif in the polypeptide to obtain GPBP resulted in anotable increase in polypeptide interaction. All of the above dataindicate that GPBPΔ26 self-associates in vivo, and that the insertion ofthe 26-residues into the polypeptide chain yields a more interactivemolecular species.

GPBP is highly expressed in human but not in bovine and murineglomerulus and alveolus. We have shown that GPBP/GPBPΔ26 ispreferentially expressed in human cells and tissues that are commonlytargeted in naturally occurring autoimmune responses. To specificallyinvestigate the expression of GPBP, we raised polyclonal antibodiesagainst a synthetic peptide representing the 26-residue motifcharacteristic of this kinase isoform, and used it forimmunohistochemical studies on frozen or formalin fixed paraffinembedded human tissues (FIG. 13). In general, these antibodies showedmore specificity than the antibodies recognizing both isoforms for thetissue structures that are target of autoimmune responses such as thebiliary ducts, the Langerhans islets or the white matter of the centralnervous system (not shown). Nevertheless, the most remarkable findingwas the presence of linear deposits of GPBP-selective antibodies aroundthe small vessels in every tissue studied (A), suggesting that GPBP isassociated with endothelial basement membranes. Consequently, at theglomerulus, the anti-GPBP antibodies displayed a vascular patternclosely resembling the glomerular basement membrane staining yieldedeither by monoclonal antibodies specifically recognizing the α3(IV)NC1(compare 13B with 13C and 13D), or by circulating GP autoantibodies(compare 13E and 13F). These observations further supported the initialobservation that GPBP is expressed in tissue structures targeted innatural autoimmune responses, suggesting that the expression of GPBP isa risk factor and makes the host tissue vulnerable to an autoimmuneattack.

To further assess this hypothesis, we investigated the presence of GPBPand GPBPΔ26 in the glomerulus of two mammals that naturally do notundergo GP disease compared to human (FIG. 14). GPBP-specific antibodiesfailed to stain the glomerulus of both bovine or murine specimens(compare 14A with 14B and 14C) while antibodies recognizing theN-terminal sequence common to both GPBP and GPBPΔ26 stained thesestructures in all three species, although with different distributionsand intensities (14D-14F). In bovine renal cortex, GPBPΔ26 was expressedat a lower rate than in human, but showed similar tissue distribution.In murine samples, however, GPBPΔ26 displayed a tissue distributionclosely resembling that of GPBP in human glomerulus. Similar resultswere obtained when studying the alveolus in the three different species(not shown). To rule out that the differences in antibody detection wasdue to primary structure differences rather than to a differentialexpression, we determined the corresponding primary structures in thesetwo species by cDNA sequencing. Bovine and mouse GPBP (SEQ ID NOS:3-6and 9-12) displayed an overall identity with human material of 97.9% and96.6% respectively. Furthermore, the mouse 26-residue motif wasidentical to human while bovine diverged only in one residue. Finally,and similarly to human, we successfully amplified GPBP cDNA from mouseor bovine kidney total RNA using oligonucleotides specific for thecorresponding 78-bp exons, indicating that GPBP is expressed at very lowlevels not detectable by immunochemical techniques.

GPBP is highly expressed in several autoimmune conditions. We analyzedseveral tissues from different GP patients by specific RT-PCR to assessGPBP/GPBPΔ26 mRNA levels. As in control kidneys, the major expressedisoform in GP kidneys was GPBPΔ26. However, in the muscle of one of thepatients, GPBP was preferentially expressed, whereas GPBPΔ26 was theonly isoform detected in control muscle samples (FIG. 15 A). Since wedid not have kidney samples from this particular patient, we could notassess GPBP/GPBPΔ26 expression in the corresponding target organ. Forsimilar reasons, we could not assess GPBP/GPBPΔ26 levels in the muscleof the patients in which kidneys were studied. Muscle cells express highlevels of GPBP/GPBPΔ26 (see Northern blot in FIG. 9), and they comprisethe bulk of the tissue. In contrast, the expression of GPBP/GPBPΔ26 inthe kidney was much less, and the glomerulus was virtually the onlykidney structure expressing the GPBP isoform (see FIG. 13). Theglomerulus is a relatively less abundant structure in kidney than themyocyte is in muscle, and the glomerulus is the structure targeted byimmune attack in GP pathogenesis. These factors, together with thepreferential amplification of the more abundant and shorter messageswhen performing RT-PCR studies, could account for the lack of detectionof GPBP in both normal and GP kidneys, thus precluding the assessment ofGPBP expression at the glomerulus during pathogenesis. Nevertheless, theincreased levels of GPBP in a GP patient suggest that GPBP/GPBPΔ26expression is altered during GP pathogenesis, and that augmented GPBPexpression has a pathogenic significance in GP disease.

To investigate the expression of GPBP and GPBPΔ26 in autoimnunepathogenesis, we studied cutaneous autoimmune processes and comparedthem with control samples representing normal skin or non-autoimmunedermatitis (FIG. 15). Control samples displayed a limited expression ofGPBP in the most peripheral keratinocytes (15B, 15E), whilekeratinocytes expanding from stratum basale to corneum expressedabundant GPBP in skin affected by systemic lupus erythematosus (SLE)(15C, 15F) or lichen planus (15D, 15G). GPBP was preferentiallyexpressed in cell surface structures that closely resembled the blebspreviously described in cultured keratinocytes upon UV irradiation andapoptosis induction (6). In contrast, antibodies recognizing both GPBPand GPBPΔ26 yielded a diffuse cytosolic pattern through the wholeepidermis in both autoimmune affected or control samples (not shown).These data indicate that in both control and autoimmune-affectedkeratinocytes, GPBPΔ26 was expressed at the cytosol and that theexpression did not significantly vary during cell differentiation. Incontrast, mature keratinocytes were virtually the only GPBP expressingcells. However, bleb formation and expression of GPBP was observed inthe early stages of differentiation in epidermis affected by autoimmuneresponses (15C, 15D, 15F, 15G). This further supports previousobservations indicating that aberrant apoptosis at the basalkeratinocytes is involved in the pathogenesis of autoimmune processesaffecting skin (7), and suggests that apoptosis and GPBP expression arelinked in this human cell system.

Discussion

Alternative pre-mRNA splicing is a fundamental mechanism fordifferential gene expression that has been reported to regulate thetissue distribution, intracellular localization, and function ofdifferent protein kinases (8-11). In this regard, and closely resemblingGPBP, B-Raf exists as multiple spliced variants, in which the presenceof specific exons renders more interactive, efficient and oncogenickinases (12).

Although it is evident that rGPBPΔ26 still bears the uncharacterizedcatalytic domain of this novel kinase, both auto- andtrans-phosphorylating activities are greatly reduced when compared torGPBP. Gel filtration and two hybrid experiments provide some insightsinto the mechanisms that underlie such a reduced phosphate transferactivity. About 1-2% of rGPBP is organized in very high molecular weightaggregates that display about one third of the phosphorylating activityof rGPBP, indicating that high molecular aggregation renders moreefficient quaternary structures. Recombinant GPBPΔ26, with virtually nopeak I material, consistently displayed a reduced kinase activity.However, aggregation does not seem to be the only mechanism by which the26-residues increases specific activity, since the rGPBPΔ26 materialpresent in peak II also shows a reduced phosphorylating activity whencompared to homologous fractions of rGPBP. One possibility is thatrGPBP-derived aggregates display higher specific activities because ofquaternary structure strengthening caused by the insertion of the26-residue motif. The oligomers are kept together mainly by very strongnon-covalent bonds, since the bulk of the material appears as a singlepolypeptide in non-reducing SDS-PAGE, and the presence of either 8 Murea or 6 M guanidine had little effect on chromatographic gelfiltration profiles (not shown). How the 26-residue motif renders a morestrengthened and active structure remains to be clarified.Conformational changes induced by the presence of an exon encoded motifthat alter the activation status of the kinase have been proposed forthe linker domain of the Src protein (24) and exons 8b and 10 of B-Raf(12). Alternatively, the 26-residue motif may provide the structuralrequirements such as residues whose phosphorylation may be necessary forfull activation of GPBP.

We have reported (13) that the primary structure of the GP antigen(α3(IV)NC1) is the target of a complex folding process yielding multipleconformers. Isolated conformers are non-minimum energy structuresspecifically activated by phosphorylation for supramolecular aggregationand likely quaternary structure formation. In GP patients, the α3(IV)NC1shows conformational alterations and a reduced ability to mediate thedisulfide stabilization of the collagen IV network. The GP antibodies,in turn, demonstrate stronger affinity towards the patient α3(IV)NC1conformers, indicating that conformationally altered material caused theautoimmune response. Therefore, it seems that in GP disease an earlyalteration in the conforming process of the α3(IV)NC1 could generatealtered conformers for which the immune system is not tolerant, thusmediating the autoimmune response.

Other evidence (Raya et al., unpublished results) indicates thatphosphorylation is the signal that drives the folding of the α3(IV)NC1into non-minimum energy ends. In this scenario, three features of thehuman α3(IV)NC1 system are of special pathogenic relevance when comparedto the corresponding antigen systems from species that, like bovine ormurine, do not undergo spontaneous GP disease. First, the N-terminus ofthe human α3(IV)NC1 contains a motif that is phosphorylatable by PKA andalso by GPBP (see above, and also 2-4). Second, the human gene generatesmultiples alternative products by alternative exon splicing (14,15).Exon skipping generates alternative products with divergent C-terminalends that up-regulate the in vitro PKA phosphorylation of the primaryα3(IV)NC1 product (See below Example 3). Third, the human GPBP isexpressed associated with glomerular and alveolar basement membranes,the two main targets in GP disease. The phosphorylation-dependentconforming process is also a feature of non-pathogenic NC1 domains (13),suggesting that the phosphorylatable N-terminus, the alternativesplicing diversification, and the expression of GPBP at the glomerularand alveolar basement membranes, are all exclusively human features thatplace the conformation process of α3(IV)NC1 in a vulnerable condition.The four independent GP kidneys studied expressed higher levels of GPantigen alternative products (15; Bernal and Saus, unpublished results),and an augmented expression of GPBP were found in a GP patient (seeabove). Both increased levels of alternative GP antigen products andGPBP are expected to have consequences in the phosphorylation-dependentconformational process of the α3(IV)NC1, and therefore with pathogenicpotential.

GPBP is highly expressed in skin targeted by natural autoimmuneresponses. In the epidermis, GPBP is associated with cell surface blebscharacteristic of the apoptosis-mediated differentiation process thatkeratinocytes undergo during maturation from basale to corneum strata(22, 23). Keratinocytes from SLE patients show a remarkably heightenedsensitivity to UV-induced apoptosis (6, 18, 20), and augmented andpremature apoptosis of keratinocytes has been reported to exist in SLEand dermatomyositis (7). Consistently, we found apoptotic bodiesexpanding from basal to peripheral strata of the epidermis in severalskin autoimmune conditions including discoid lupus (not shown), SLE andlichen planus. Autoantigens, and modified versions thereof are clusteredin the cell surface blebs of apoptotic keratinocytes (6,18,20).Apoptotic surface blebs present autoantigens (21), and likely releasemodified versions to the circulation (16-20). It has been suggested thatthe release of modified autoantigens from apoptotic bodies could be theimmunizing event that mediates systemic autoimmune responses mediatingSLE and scleroderma (18,19).

Our evidence indicates that both GPBP and GPBPΔ26 are able to act invitro as protein kinases, with GPBP being a more active isoform thanGPBPΔ26. Furthermore, recombinant material representing GPBP or GPBPΔ26purified from yeast or from human 293 cells contained an associatedproteolytic activity that specifically degrades the α3(IV)NC1 domain(unpublished results). The proteolytic activity operates on α3(IV)NC1produced in an eukaryotic expression system, but not on recombinantmaterial produced in bacteria (unpublished results), indicating thatα3(IV)NC1 processing has some conformational or post-translationalrequirements not present in prokaryotic recombinant material. Finally,it has been reported that several autoantigens undergo phosphorylationand degradation in apoptotic keratinocytes (20). While not being limitedto an exact mechanism, we propose, in light of all of the above data,that the machinery assembling GPBP at the apoptotic blebs likelyperforms a complex modification of the autoantigens that includesphosphorylation, conformational changes and degradation. Accordingly,recombinant protein representing autoantigens in SLE (P1 ribosomalphosphoprotein and Sm-D1 small nuclear ribonucleoproteins) and indermatomyositis (hystidil-tRNA synthetase) were in vitro substrates ofGPBP (unpublished results).

The down-regulation in cancer cell lines of GPBP, suggest that the cellmachinery harboring GPBP/GPBPΔ26 is likely involved in signalingpathways inducing programmed cell death. The corresponding apoptoticpathway could be up regulated during autoimmune pathogenesis to cause analtered antigen presentation in individuals carrying specific MHChaplotypes; and down regulated during cell transformation to preventautoimmune attack to the transformed cells during tumor growth.

References for Example 2

-   1. Saus, J. (1998) in Goodpasture's Syndrome: Encyclopedia of    Immunology 2^(nd) edn. Vol. 2, eds. Delves, P. J., & Roitt, I. M.,    (Academic Press Ltd., London), pp. 1005-1011.-   2. Quinones, S., Bernal, D., García-Sogo, M., Elena S. F., &    Saus, J. (1992) J. Biol. Chem. 267, 19780-19784.-   3. Revert, F., Penadés, J. R., Plana, M., Bernal, D., Johansson, C.,    Itarte, E., Cervera, J., Wieslander, J., Quinones, S., &    Saus, J. (1995) J. Biol. Chem. 270, 13254-13261.-   4. Raya, A., Revert, F., Navarro, S., & Saus, J. (1999) J. Biol.    Chem. 274, 12642-12649.-   5. Green, M. R. (1986) Ann. Rev. Genet. 20, 671-708.-   6. Casciola-Rosen, L. A., Anhalt, G. & Rosen, A. (1994) J. Exp. Med.    179:1317-1330.-   7. Pablos, J. L:, Santiago, B., Galindo, M., Carreira, P. E.,    Ballestin, C.& Gomez-Reino, J. J. (1999) J. Pathol. 188: 63-68.-   8. Srinivasan, M., Edman, C. F., & Schulman, H. (1994) J. Cell.    Biol. 126, 839-852.-   9. Naito, Y., Watanabe, Y., Yokokura, H., Sugita, R., Nishio, M., &    Hidaka, H. (1997) J. Biol. Chem. 272, 32704-32708.-   10. Bayer, K.-U., Löhler, J., & Harbers, K. (1996) Mol. Cell. Biol.    16, 29-36.-   11. Madaule, P., Eda, M., Watanabe, N, Fujisawa, K., Matsuoka, T.,    Bito, H., Ishizaki, T., & Narumiya, S. (1998) Nature 394, 491-494.-   12. Papin, C., Denouel-Galy, A., Laugier, D., Calothy, G., &    Eychène, A. (1998) J. Biol. Chem. 273, 24939-24947.-   13. U.S. Provisional Patent Application, Ser. No. to be assigned,    filed Feb. 11, 2000 (Case number 98,723-C)-   14. Penadés, J. R., Bernal, D., Revert, F., Johansson, C.,    Fresquet, V. J., Cervera, J., Wieslander, J., Quinones, S. &    Saus, J. (1995) Eur. J. Biochem. 229, 754-760.-   15. Bernal, D., Quinones, S., & Saus, J. (1993) J. Biol. Chem., 268,    12090-12094.-   16. Casciola-Rosen, L. A., Anhalt, G. J. & Rosen, A. (1995) J. Exp.    Med. 182: 1625-1634.-   17. Casiano, C. A., Martin, S. J., Green, D. R., &    Tan, E. M. (1996) J. Exp. Med. 184: 765-770.-   18. Casciola-Rosen, L., & Rosen, A. (1997) Lupus 6: 175-180.-   19. Bolívar, J., Guelman, S., Iglesias, C., Ortíz, M., &    Valdivia, M. (1998) J. Biol. Chem. 273: 17122-17127.-   20. Utz, P. J., & Anderson, P. (1998) Arthritis Rheum. 41:    1152-1160.-   21. Golan, T. D., Elkon, K. B., Ghavari, A. E.,& Krueger, J.    G.(1992) J. Clin. Invest. 90: 1067-1076.-   22. Polalowska, R. R., Piacentini, M., Bartlett, R., Goldsmith, L.    A., & Haake, A. R. (1994) Dev. Dinam. 199: 176-188.-   23. Maruoka, Y., Harada, H., Mitsuyasu et al. (1997) Biochem.    Biophys. Res. Commun. 238: 886-890.-   24. Xu, W., Harrison, S. C., & Eck, M. J. (1997) Nature 385,    595-602.

EXAMPLE 3 Regulation of Human Autoantigen Phosphorylation by ExonSplicing

Introduction

In GP disease, the immune system attack is mediated by autoantibodiesagainst the non-collagenous C-terminal domain (NC1) of the α3 chain ofcollagen IV (the GP antigen) (1). The N-terminus of the human α3(IV)NC 1contains a highly divergent and hydrophilic region with a uniquestructural motif, KRGDS⁹, that harbors a cell adhesion signal as anintegral part of a functional phosphorylation site for type A proteinkinases (2,3). Furthermore, the gene region encoding the human GPantigen characteristically generates multiple mRNAs by alternative exonsplicing (4,5). The alternative products diverge in the C-terminal endsand all but one share the N-terminal KRGDS⁹ (4,5).

Multiple sclerosis (MS) is an exclusive human neurological diseasecharacterized by the presence of inflamatory demyelization plaques atthe central nervous system. (6). Several evidences indicate that thisdisease is caused by an autoimmune attack mediated by cytotoxic T cellstowards specific components of the white matter including the myelinbasic protein (MBP) (7, 8). In humans, the MBP gene generates fourproducts (MBP, MBPΔII, MBPΔV and MBPΔII/V) that result from alternativeexon splicing during pre-mRNA processing (9). Among these, MBPΔII is themore abundant form in the mature central nervous system, while MBP formcontaining all the exons is virtually absent (9).

Several biological similarities exist between the autoimme responsesmediating GP disease and MS, namely: 1) both are human exclusivediseases and typically initiate after a viral flu-like disease; 2) astrong linkage exists to the same haplotype of the HLA-DR region of theclass II MHC; 3) several products are generated by alternative splicing;and 4) the death of a MS patient by GP disease has recently beenreported (10).

Materials and Methods

Synthetic polymers: GPΔIII derived peptide, QRAHGQDLDALFVKVLRSP (SEQ IDNO:43) and GPΔIII/IV/V derived peptide, QRAHGQDLESLFHQL (SEQ ID NO:44)were synthesized using either Boc-(MedProbe) or Fmoc-(Chiron, Lipotec)chemistry.

Plasmid Construction and Recombinant Expression.

GP derived material: The constructs representing the differentGP-spliced forms were obtained by subcloning the cDNAs used elsewhere toexpress the corresponding recombinant proteins (5) into the BamHI siteof a modified pET15b vector, in which the extraneous vector-derivedamino-terminal sequence except for the initiation Met was eliminated.The extra sequence was removed by cutting the vector with NcoI and BamHI, filling-in of the free ends with Klenow, and re-ligation. Thisresulted in the reformation of both restriction sites and placed theBamHII site immediately downstream of the codon for the amino-terminalMet.

The recombinant proteins representing GP or GPAV (SEQ ID NO:46) werepurified by precipitation (5). Bacterial pellets containing therecombinant proteins representing GPΔIII (SEQ ID NO:48) or GPΔIII/IV/V(SEQ ID NO:50) were dissolved by 8 M urea in 40 mM Tris-HCl pH 6.8 andsonication. After centrifugation at 40,000×g the supernatants werepassed through a 0.22 μm filter and applied to resource Q column forFPLC. The effluent was acidified to pH 6 with HCl and applied to aresource S column previously equilibrated with 40 mM MES pH 6 for asecond FPLC purification. The material in the resulting effluent wasused for in vitro phosphorylation.

MBP-derived material: cDNA representing human MBPΔII (SEQ ID NO:51) wasobtained by RT-PCR using total RNA from central nervous system. The cDNArepresenting human MBP was a generous gift from C. Campagnoni (UCLA).Both fragments were cloned into a modified version of pHIL-D2(Invitrogen) containing a 6×His-coding sequence at the C-terminus togenerate pHIL-MBPΔII-His and pHIL-MBP-His, respectively. These plasmidswere used for recombinant expression in Pichia pastoris as described in(12). Recombinant proteins were purified using immobilized metalaffinity chromatography (TALON resin, CLONTECH) under denaturantconditions (8M urea) and eluted with 300 mM imidazole followingmanufacturers' instructions. The affinity-purified material was thenrenatured by dilution into 80 volumes of 50 mM Tris-HCl pH 8.0, 10 mMCHAPS, 400 mM NaCl, 2 mM DTT, and concentrated 50 times byultrafiltration through a YM10-type membrane (AMICON). The Ser to Alamutants were produced by site-directed mutagenesis over nativesequence-containing constructs using transformer mutagenesis kit fromCLONTECH and the resulting proteins were similarly produced.

Phosphorylation studies. Phosphorylation studies were essentially doneas described above (see also 3 and 12). In some experiments, thesubstrates were in-blot renatured and then, phosphorylated for 30 min atroom temperature by overlaying 100 μl of phosphorylation buffercontaining 0.5 μg of rGPBP. Digestion with V8 endopeptidase andimmunoprecipitation were performed as described in (3).

Antibody production. Synthetic peptides representing the C-terminaldivergent ends of GPΔIII or GPΔIII/IV/V comprised in SEQ ID NO:43 or SEQID NO:44 respectively were conjugated to a cytochrome C, BSA orovoalbumine using a glutaraldehyde coupling standard procedure. Theresulting protein conjugates were used for mouse immmunization to obtainpolyclonal antibodies specific for GPΔIII and monoclonal antibodiesspecific for GPΔIII/IV/V (Mab153). To obtain monoclonal antibodiesspecific for GPAV (Mab5A) mouse were immunized using recombinantbacterial protein representing the corresponding alternative formcomprising the SEQ ID NO:50. The production of monoclonal (M3/1, P1/2)or polyclonal (anti-GPpep1) antibodies against SEQ ID NO: 26 whichrepresents the N-terminal region of the GP alternative forms have beenpreviously described (3,5).

Boc-based Peptide Synthesis.

Assembling The peptide was assembled by stepwise solid phase synthesisusing a Boc-Benzyl strategy. The starting resin used was Boc-Pro-PAMresin (0.56 meq/g, batch R4108). The deprotection/coupling procedureused was: TFA (×1 min) TFA (1×3 min) DCM (flow flash) Isopropylalcohol(1×30 sec) DMF (3×1 min) COUPLING/DMF (1×10 min) DMF (1×1min)COUPLING/DMF (1×10 min) DMF (2×1 min) DCM (1×1 min). For each step 10 mlper gram of peptide-resin were used. The coupling of all amino acids(fivefold excess) was performed in DMF in the presence of BOP, Hobt andDIEA. For the synthesis the following side-chain protecting groups wereused: benzyl for serine; 2 chlorobenzyloxycarbonyl for lysine;cyclohexyl for aspartic and glutamic acid; tosyl for histidine andarginine.

Cleavage. The peptide was cleaved from the resin and fully deprotectedby a treatment with liquid Hydrogen Fluoride (HF): Ten milliliters of HFper gram of peptide resin were added and the mixture kept at 0° C. for45 min in the presence of p-cresol as scavengers. After evaporation ofthe HF, the crude reaction mixture is washed with ether, dissolved inTFA, precipitated with ether and dried.

Purification. Stationary phase: Silica C18, 15 μm, 120 A; Mobile phase:solvent A: water 0.1% TFA and solvent B: acetonitrile/A, 60/40 (v/v);Gradient: linear from 20 to 60% B in 30 min; Flow rate: 40 ml/min; anddetection was U.V (210 nm). Fractions with a purity higher than 80% werepooled and lyophilized. Control of purity and identity was performed byanalytical HPLC and ES/MS. The final product had 88% purity and anexperimental molecular weight of 2192.9.

Fmoc-based Peptide Synthesis.

Assembling The peptides were synthesized by stepwise linear solid phaseon Pro-clorotrityl-resin (0.685 meq/g) with standard Fmoc/tBu chemistry.The deprotection/coupling procedure used was: Fmoc aa (0.66 g) HOBt(0.26 g) DIPCDI (0.28 ml) for 40 min following a control by Kaiser test.If the test was positive the time was extended until change to negative.Then DMF (31 min), piperidine/DMF 20% (11 min) piperidine/DMF 20% (15min) and DMF (41 min). Side chain protectors were: Pmc(pentamethylcromane sulfonyl) for arginine, Bcc (tert-butoxycarbonyl)for lysine, tBu (tert-butyl) for aspartic acid and for serine and Trl(trityl) for histidine.

Cleavage. The peptide was cleaved and fully deprotected by treatmentcleavage with TFA/water 90/10. Ten milliliters of TFA solution per gramof resin were added. Water acts as scavenger. After two hours, resin wasfiltered and the resulting solution was precipitated five times withcold diethylether. The final precipitated was dried.

Purification. Stationary phase: Kromasil C18 10 μm; Mobile phase:solvent A: water 0.1% TFA and solvent B: acetonitrile 0.1% TFA;Isocratic: 28% B; Flow rate: 55 ml/min; Detection: 220 nm. Fractionswith the higher purity were pooled and lyophilized, and a second HPLCpurification round performed. Control of purity and identity wasperformed by analytical HPLC and ES/MS. The final product had 97% purityand an experimental molecular weight of 2190.9.

Results

Regulation of the phosphorylation of the human GP antigen by alternativesplicing. We produced bacterial recombinant proteins representing theprimary antigen (GP) or the individual alternative products GPΔV (SEQ IDNO:46), GPΔIII (SEQ ID NO:48) and GPΔIII/IV/V (SEQ ID NO:50), and wetested their ability to be phosphorylated by PKA (FIG. 16, left panel ).Using standard ATP concentrations (150 μM), all four recombinantantigens were phosphorylated but to very different extents. Thealternative forms incorporated ³²p more efficiently than the primary GPantigen, suggesting that they are better substrates. Because theseantigens are expected to be in the extracellular compartment, we alsoassayed their phosphorylatability with more physiological ATPconcentrations (0.1-0.5 μM). Under these conditions, the differences in³²P incorporation between the primary and alternative products were moreevident, indicating that at low ATP concentrations the primary GPantigen was a very poor substrate for the kinase. Among the three PKAphosphorylation sites present in the GP antigen, the N-terminal Ser⁹ andSer²⁶ are the major ones, and are common to all the alternative productsassayed (3,5). Accordingly, the differences observed in phosphorylationfor the full polypeptides also existed among the individual N-terminalregions, as determined after specific V8 digestion andimmunoprecipitation (not shown). This strongly suggests that differencesin phosphorylation might be due to the presence of different C-terminalsequences in the alternative products. Since GPΔIII and GPΔIII/IV/Vdisplayed significantly higher ³²P incorporation rates than GPΔV, andthey have shorter divergent C-terminal regions (5), we used syntheticpeptides individually representing these C-terminal sequences (SEQ IDNO: 43, SEQ ID NO:44) to further examine their regulatory roles in thein vitro phosphorylation of the native antigen. Collagen IV is atrimeric molecule comprised of three interwoven α chains. In basementmembranes, two collagen IV molecules assemble through their NC1 domainsto yield a hexameric NC1 structure that can be solubilized by bacterialcollagenase digestion (1). Dissociation of the hexamer structurereleases the GP antigen in monomeric and disulfide-related dimeric forms(1). For the following set of experiments, we carried outphosphorylations in the presence of low, extracellular-like ATPconcentrations using both monomeric or hexameric native GP antigen (FIG.16, right panel ). The presence of each specific peptide but not controlpeptides (not shown) induced the phosphorylation of a single polypeptidedisplaying an apparent MW of 22 kDa. By specific V8 digestion andimmunoprecipitation, the corresponding polypeptide has been identifiedas the 22 kDa conformer of the α3(IV)NC1, previously characterized andidentified as the best substrate for the PKA (11).

Regulation of the phosphorylation of the MBP by alternative splicing.The MBP contains at its N terminal region two PKA phosphorylation sites(Ser⁸, Ser⁵⁷) that are structurally similar to the N terminus site(Ser⁹) present in GP antigen products (FIG. 17). The Ser⁸ site presentin all the MBP proteins is located in a similar position than the Ser⁹in the GP-derived polypeptides. In addition, in the MBP and GPΔIII Ser⁸and Ser⁹ respectively are at a similar distance in the primarystructures of a highly homologous motif present in the correspondingexon II (bend arrow in FIG. 17). The GPΔIII-derived motif coincides withthe C terminal divergent region that up-regulates PKA phosphorylation ofSer⁹ in the GP antigen system (FIG. 16). The regulatory-like sequence inMBP is located at exon II and its presence in the final products dependson an alternative exon splicing mechanism. Therefore, the MBP motifidentified by structural comparison to GPΔIII may be also regulating PKAphosphorylation of Ser⁸. We produced recombinant proteins representingMBP and MBPΔII (SEQ ID NO:54) and the corresponding Ser to Ala mutantsto knock-out each of the two PKA phosphorylation sites (Ser⁸ and Ser⁵⁷)present in exon I. Subsequently, we assessed its in vitrophosphorylation by PKA (FIG. 18). MBPΔII was a better substrate thanMBP, and Ser⁸ was the major phosphorylation site, indicating that,similarly to GP antigenic system, alternative exon splicing regulatesthe PKA phosphorylation of specific sites located at the N-terminalregion common to all the MBP-derived alternative forms.

In similar experiments assessing GPBP phosphorylation of the recombinantMBP proteins, GPBP preferentially phosphorylated MBP, while littlephosphorylation of MBPΔII was observed (FIG. 19). Furthermore,recombinant Ser to Ala mutants displayed no significant reduction in ³²Pincorporation, indicating that GPBP phosphorylates MBP/MBPΔII in anopposite way than PKA, and that these two kinases do not share majorphosphorylation sites in MBP proteins.

From all these data we concluded that in the MBP system, alternativesplicing regulates the phosphorylation of specific serines by either PKAor GPBP.

Synthetic peptides representing the C terminal region of GPΔIIIinfluence GPBP phosphorylation. To assess the effect of the C terminalregion of GPΔIII on GPBP activity, peptides representing this regionwere synthesized using two different chemistries (Boc or Fmoc), andseparately added to a phosphorylation mixture containing GPBP (FIG. 20).Boc-based synthetic peptides positively influenced GPBPautophosphorylation while Fmoc-based inhibited GPBP autophosphorylation,suggesting that the regulatory sequences derived from the alternativeproducts in either GP and MBP antigenic systems can influence the kinaseactivity of GPBP.

Discussion

We have shown that the α3(IV)NC1 domain undergoes a complex structuraldiversification by two different mechanism: 1) alternative splicing(4,5) and 2) conformational isomerization of the primary product (11).Both mechanisms generate products that are distinguished by PKA,indicating that PKA phosphorylation is a critical event in the biologyof the α3(IV)NC1 domain. Phosphorylation guides at least in part thefolding, but also the supramolecular assembly of the α3(IV)NC1 domain inthe collagen IV network (11 and Raya et al. unpublished results).Altered conformers of the α3(IV)NC1 lead the autoimmune responsemediating GP disease (11), suggesting that an alteration in antigenphosphorylation could be the primary event in the onset of the disease.Accordingly, we have found increased expression levels of GPΔIII inseveral GP kidneys (4 and Bernal and Saus, unpublished results), and anincreased expression of GPBP has been detected in another Goodpasturepatient (FIG. 15). Both increased expression of alternative GP antigenproducts and of GPBP are expected to have consequences in thephosphorylation steady state of α3(IV)NC1, and therefore in thecorresponding conformational process. The discrimination among thedifferent structural products by PKA strongly suggests that this kinase,or another structurally similar kinase, is involved in the physiologicalantigen conforming process, and that antigen phosphorylation by GPBP hasa pathogenic significance. In pathogenesis, GPBP could be an intrudingkinase, interfering in the phosphorylation-dependent conforming process.Accordingly, GPBP is expressed in tissue structures that are targeted bynatural autoimmune responses, and an increased expression of GPBP isassociated with several autoimmune conditions (See examples 1 and 2above).

An alternative splicing mechanism also regulates the PKA phosphorylationof specific serines in the MBP antigenic system. MBP is also a substratefor GPBP suggesting that GPBP may play a pathogenic role in multiplesclerosis, and other autoimmune responses.

All of the above data identify GPBP as a potential target fortherapeutics in autoimmune disease. In FIG. 20, we show that syntheticpeptides representing the C terminal region of GPΔIII (SEQ ID NO:43)modulate the action of GPBP in vitro, and therefore we identified thisand related sequences as peptide-based compounds to modulate theactivity of GPBP in vivo. The induction of GP antigen phosphorylation byPKA was achieved when using Boc-based peptides, but not when usingsimilar Fmoc-based peptides. Furthermore, Boc- but not Fmoc-basedpeptides were in vitro substrates of PKA (not shown), indicating thatimportant structural differences exist between both products. Since bothproducts displayed no significant differences in mass spectrometry, onepossibility is that the different deprotection procedure used may beresponsible for conformational differences in the secondary structurethat may be critical for biological activity. Accordingly, Boc-basedpeptide loses its ability to induce PKA upon long storage at lowtemperatures.

REFERENCES FOR EXAMPLE 3

-   1. Saus, J. (1998) in Goodpasture's Syndrome: Encyclopedia of    Immunology 2^(nd) edn. Vol. 2, eds. Delves, P. J., & Roitt, I. M.,    (Academic Press Ltd., London), pp. 1005-1011.-   2. Quinones, S., Bernal, D., García-Sogo, M., Elena S. F., &    Saus, J. (1992) J. Biol. Chem. 267, 19780-19784.-   3. Revert, F., Penadés, J. R., Plana, M., Bernal, D., Johansson, C.,    Itarte, E., Cervera, J., Wieslander, J., Quinones, S., &    Saus, J. (1995) J. Biol. Chem. 270, 13254-13261.-   4. Bernal, D., Quinones, S., & Saus, J. (1993) J. Biol. Chem., 268,    12090-12094.-   5. Penadés, J. R., Bernal, D., Revert, F., Johansson, C.,    Fresquet, V. J., Cervera, J., Wieslander, J., Quinones, S. &    Saus, J. (1995) Eur. J. Biochem. 229, 754-760.-   6. Raus, J. C M, en Multiple Sclerosis: Encyclopedia of Immunology    2^(nd) edn. Vol. 3 (eds.

Delves, P. J., & Roitt, I. M.) 1786-1789 (Academic Press Ltd., London,1998). 7. Pette, M., Fujita, K., Wilkinson, D., Altmann, D. M.,Trowsdale, J., Giegerich, G., Hinkkanen, A., Epplen, J. T., Kappos, L.,and Wekerle, H. (1994) Proc. Natl. Acad Sci. USA 87, 7968-7972

-   8. Tschida, T., Parker, K. C., Turner, R. V., McFarland, H. F.,    Coligan, J. E., and Biddison, W. E. (1994) Proc. Natl. Acad. Sci.    USA 91, 10859-10863.-   9. Campagnoni, A. T. (1988) J. Neurochem. 51, 1-14.-   10. Henderson, R. D., Saltissi, D., and Pender, M. P. (1998) Acta    Neurol. Scand 98, 134-135.-   11. U.S. Provisional Patent Application, Ser. No. to be assigned,    filed Feb. 11, 2000 (Case number 98, 723-C).-   12. Raya, A., Revert, F., Navarro, S., and Saus, J. (1999). J. Biol.    Chem. 274, 12642-12649.

The present invention is not limited by the aforementioned particularpreferred embodiments. It will occur to those ordinarily skilled in theart that various modifications may be made to the disclosed preferredembodiments without diverting from the concept of the invention. Allsuch modifications are intended to be within the scope of the presentinvention.

1. An isolated nucleic acid sequence comprising a sequence substantiallysimilar to a nucleic acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, and SEQ ID NO:25.
 2. A recombinant expressionvector comprising the isolated nucleic acid sequence of claim
 1. 3. Ahost cell transfected with the recombinant expression vector of claim 2.4. A substantially purified polypeptide, comprising an amino acidsequence substantially similar to a sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, or peptide fragments thereof
 5. Anantibody that selectively binds to the substantially purified protein orpolypeptide of claim
 4. 6. A method for detecting the presence of aprotein that is substantially similar to a protein selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, comprising a) providing aprotein sample to be screened; b) contacting the protein sample to bescreened with the antibody of claim 5 under conditions that promoteantibody-antigen complex formation; and c) detecting the formation ofantibody-antigen complexes, wherein the presence of the antibody-antigencomplex indicates the presence of a protein that is substantiallysimilar to a protein selected from the group consisting of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24.
 7. The method of claim 6, wherein the method is used to detect anautoimmune condition in a patient.
 8. The method of claim 6, wherein themethod is used to detect cells undergoing apoptosis or cancertransformation in a patient.
 9. A method for detecting in a sample asequence that is substantially similar to a nucleic acid selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25,comprising contacting the sample with the isolated nucleic acid of claim1, or fragments thereof, and detecting complex formation, whereincomplex formation indicates the presence in the sample of the sequencethat is substantially similar to a nucleic acid selected from the groupconsisting of SEQ ID) NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25.
 10. The method ofclaim 9, wherein the method is used to detect an autoimmune condition ina patient.
 11. The method of claim 9, wherein the method is used todetect cells undergoing apoptosis or cancer transformation in a patient.12. A method for treating a patient with an autoimmune disorder and/or atumor, comprising modifying the expression or activity of GPBP, GPBPΔ26,or a protein comprising a polypeptide substantially similarly toGPBPpep1 in the patient with the autoimmune disorder and/or tumor.